GSDMD fusion protein with controllable perforation activity outside cells and application of GSDMD fusion protein

Abstract

The invention discloses a GSDMD fusion protein with controllable perforation activity outside cells and application of the GSDMD fusion protein. A GSDMD protein in a self-inhibition state is connected with a polypeptide fragment of a targeted recognition tissue or cell, the formed fusion protein can be specifically combined and gathered to the surface of a target cell outside the cell, and the GSDMD protein contained in the fusion protein is converted into an active state from an inactive state under the condition that the GSDMD protein is not cut by using a GSDMD direct agonist, so that the fusion protein can be specifically combined and gathered to the surface of the target cell. Then, cell membrane pore-forming of tumor cells is performed to induce pyroptosis of the cells, and toxic and side effects caused by medication of a small molecule drug DMB can be effectively reduced. In vitro experiments show that the composition and the use method provided by the invention have a good killing effect on various tumor cell lines. The method does not depend on cell endocytosis, and does not depend on cell endogenous GSDMD protein and protease cleavage, so that the method has better targeting and killing effects on target cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to a GSDMD fusion protein with controllable perforation activity outside cells and its applications. Background Art

[0002] Pyroptosis is a programmed cell necrosis triggered by gasdermin proteins such as GSDMD located in the cytoplasm. GSDMD consists of two conserved domains: an N-terminal domain (GSDMD-N) and a C-terminal (GSDMD-C) domain. Among them, GSDMD-N is the main functional domain, while GSDMD-C has a self-inhibitory effect. When GSDMD is cleaved by inflammatory caspase proteases, the released GSDMD-N domain forms pores on the inner side of the cell membrane, leading to cell swelling, membrane perforation, and release of cell contents, thereby activating strong inflammatory and immune responses.

[0003] Existing studies have found (a patent with publication number CN118063584A) that a GSDMD variant fusion protein is formed by inserting a cathepsin D cleavage site between the N-terminal domain and the C-terminal domain of GSDMD using genetic engineering and linking it with an antibody. After this fusion protein targets a specific cell surface and is endocytosed, under the cleavage of cathepsin D in the cell, the GSDMD-N terminal domain is released, thereby forming pores in the cell membrane and inducing pyroptosis. The main problems with this method are: this fusion protein cannot exert a perforation function outside cells and must rely on the endocytosis process in cells and the cleavage action of intracellular proteases to cleave out the GSDMD-N terminal domain in cells, and the activation process and time of cell membrane perforation cannot be precisely controlled.

[0004] Existing studies have also found (literature DOI: 10.1016 / j.heliyon.2024.e30444.) that a fusion protein is composed of the HER2 antibody P1h3, an albumin-binding peptide, a cathepsin B cleavage site, an endosome-disrupting peptide E5C3, and the GSDMD-N terminal domain. This fusion protein can specifically recognize HER2-positive tumor cells and be endocytosed into cells. After entering the endosome, it is cleaved under the action of cathepsin B, and the released GSDMD-N terminal domain enters the cytoplasm during the endosome escape process mediated by E5C3, ultimately inducing pyroptosis in tumor cells. However, the main limitation of this strategy is: this fusion protein cannot exert a perforation function outside cells and still relies on the endocytosis process in cells and the endogenous cleavage action in cells to cleave out the GSDMD-N terminal domain in cells, and the activation process and time of membrane perforation cannot be precisely controlled. In addition, due to the complex design, the activity of this fusion protein is affected by many factors.

[0005] Existing studies have also found (Literature DOI: 10.1016 / j.cell.2024.08.007.) that the GSDMD small molecule agonist DMB (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline, Chinese for 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) can directly activate endogenous GSDMD in cells without cleaving GSDMD, cause perforations on the inner surface of the cell membrane, and trigger pyroptosis. The main limitations of this method are: there is no naturally occurring GSDMD outside the cell, the agonist effect of DMB is limited by intracellular GSDMD, and it cannot target specific target cells.

[0006] In summary, developing therapies that induce pyroptosis in target cells to kill tumor cells is expected to become a powerful tool against cancer, is a promising treatment strategy, and has important research significance. However, the current research on related drugs and therapies is still in the early stage. Summary of the Invention

[0007] Existing GSDMD fusion protein technologies are generally limited to: being unable to exert membrane perforation effects outside the cell, relying on endocytosis and protease cleavage, requiring the GSDMD-N terminal domain released inside the target cell, and methods for triggering cell membrane perforation and promoting pyroptosis. In view of the deficiencies in the prior art, the present invention provides a GSDMD fusion protein with controllable perforation activity outside the cell and an application solution. The GSDMD fusion protein and application solution designed in the present invention achieve the function of GSDMD cell membrane perforation outside the cell, do not rely on endocytosis, do not need to be cleaved by proteases to release the GSDMD-N terminal domain, and are controllably activated to have perforation activity at specific times and concentrations by combining with external direct agonists, which can be analogized to "switch-type" treatment, improving clinical controllability and safety, achieving precise killing and immune activation of tumor cells, and significantly superior to the limitations of the prior art.

[0008] To achieve the above objectives, the specific technical solutions of the present invention are as follows: The present invention provides a GSDMD fusion protein with controllable perforation activity outside the cell. Specifically, a human GSDMD protein polypeptide fragment (fragment A) is linked to a polypeptide fragment (fragment B) targeting the extracellular surface antigen of tumor cells to form a novel fusion protein, where: Fragment A is a polypeptide fragment at positions X-Y of the human GSDMD protein sequence, which is naturally in a self-inhibited state. Among them, X is an integer selected from 1 to 6, and Y is an integer selected from 479 to 484, including the N-terminal domain and C-terminal domain of the GSDMD protein; Fragment B is a polypeptide fragment that targets and recognizes tissues or cells.

[0009] The fusion protein described in the present invention may include one or more Fragment A and one or more Fragment B, and the present invention does not limit their repetition number and connection order. When the fragment repetition number is not 1, the sequences of two repeated fragments may be the same or different, and the present invention does not limit this. Fragment B may target the same tissue or cell, or may target different tissues or cells, and the present invention does not limit this. To maintain the integrity of the structures of Fragment A and Fragment B, a linker may or may not be included in the fusion protein, and the present invention does not limit this. In the present invention, the linker is located between two adjacent fragments, such as between Fragment A and Fragment B, or between two Fragment A, or between two Fragment B, and the present invention does not limit this.

[0010] The present invention also provides a combined application method of the GSDMD fusion protein and a GSDMD direct agonist ( Figure 1 ). The GSDMD direct agonist can directly activate the pore-forming activity of GSDMD without cleaving GSDMD. Specifically: express and prepare the GSDMD fusion protein described in the present invention. The GSDMD fusion protein can specifically target the antigen on the surface of target cells outside the cell and aggregate on the outer surface of the target cells. In this state, the fusion protein only exerts the targeting and inhibitory function on the antigen on the outer surface of tumor cells through Fragment B, but does not have the activity of membrane pore formation, reducing the cytotoxic side effects on cells. At a specified time, apply a specified concentration of the GSDMD direct agonist, controllably enabling Fragment A of the GSDMD fusion protein to be converted into a membrane pore-forming active state without being cleaved, thereby forming pores on the outer surface of the tumor cell membrane and inducing pyroptosis of the target cells, achieving the therapeutic effects of directly killing tumor cells and enhancing the anti-tumor immune response.

[0011] The present invention has found that adding the full-length GSDMD protein alone outside tumor cells, or adding the GSDMD fusion protein described in the present invention alone, cannot cause perforation of the tumor cell membrane. Further, adding a combination of the full-length GSDMD protein and the small molecule agonist DMB outside tumor cells also cannot cause perforation of the tumor cell membrane. In sharp contrast, the combined application of the GSDMD fusion protein described in the present invention and the direct agonist DMB can efficiently cause direct membrane perforation on the outer membrane of tumor cells and induce pyroptosis. Moreover, at the same concentration, the synergistic effect of the combined use of the GSDMD fusion protein and the direct agonist DMB is significantly greater than the effect of using the GSDMD fusion protein or the direct agonist DMB alone. Thus, the use concentrations of the GSDMD fusion protein and the direct agonist can be greatly reduced, the toxic side effects on non-target cells can be reduced, and the purpose of safely and controllably directly killing the target tumor cells can be achieved. The design of the present invention is simple and efficient, does not rely on endocytosis of cells and cleavage by intracellular proteases, and significantly reduces the restrictive factors for exerting activity. Further, the present invention can be used in combination with other tumor treatment regimens to enhance the anti-tumor treatment effect.

[0012] Preferably, the amino acid sequence of the human GSDMD protein is as shown in SEQ ID No.1, or a sequence shown by its variant.

[0013] Preferably, a polypeptide fragment at positions 6-479 of the human GSDMD protein sequence is selected, or a sequence shown by its variant.

[0014] Preferably, fragment B of the fusion protein is a polypeptide fragment targeting the tumor cell surface antigens EGFR, PDL1 or HER2. The polypeptide fragment is derived from the EGFRn reported in the literature, the nanobody targeting PDL1, and the genetically engineered antibody DARPin targeting HER2, and the amino acid sequence is as shown in SEQ ID No.2-4, or a sequence shown by its variant.

[0015] In multiple embodiments thereof, fragment B binds to the tumor cell surface antigen with high affinity, aggregates the fusion protein to the tumor cell surface, and still has the function of blocking the binding of the surface antigen to its ligand.

[0016] Preferably, fragment A and fragment B of the fusion protein are linked by a linker, and the amino acid sequence is as shown in SEQ ID No.8, or a sequence shown by its variant.

[0017] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No.5-7, or a sequence shown by its variant.

[0018] Among them, the variant has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity compared to the sequence from which it is derived.

[0019] The present invention also provides a composition, which comprises any one of the above-mentioned GSDMD fusion proteins and a GSDMD direct agonist, wherein the GSDMD direct agonist can directly activate the pore-forming activity of GSDMD without cleaving GSDMD.

[0020] Preferably, the GSDMD direct agonist is a small molecule compound 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline, DMB), or a DMB derivative having the effect of directly activating GSDMD. In multiple embodiments of the present invention, the working concentration of DMB is less than or equal to 5 μM, significantly less than its agonist activity for endogenous GSDMD in cells (EC 50 =5 - 10 μM), thereby effectively avoiding the influence of DMB on non-target cells and reducing cytotoxicity.

[0021] The present invention also provides biological materials related to the above-mentioned GSDMD fusion protein, and the biological materials are any one of the following: (1) A nucleic acid molecule encoding the above-mentioned GSDMD fusion protein; (2) An expression cassette containing the nucleic acid molecule described in (1); (3) A recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) A recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

[0022] Specifically: Preferably, a nucleic acid molecule can encode the above-mentioned fusion protein.

[0023] Preferably, a nucleic acid construct contains the above-mentioned nucleic acid molecule.

[0024] Preferably, an expression vector expresses the above-mentioned fusion protein, contains the above-mentioned nucleic acid molecule or contains the above-mentioned nucleic acid construct.

[0025] Preferably, a cell secretes the above fusion protein, contains the above nucleic acid molecule, contains the above nucleic acid construct or contains the above expression vector.

[0026] In one embodiment, the cell includes an immune cell.

[0027] In one embodiment, the cell includes a T cell.

[0028] In one embodiment, the cell includes a genetically engineered cell.

[0029] Preferably, a kit contains the above nucleic acid molecule, the above nucleic acid construct or the above expression vector, and is used for preparing the above fusion protein or the above cell.

[0030] Preferably, a pharmaceutical composition contains the above nucleic acid molecule, contains the above nucleic acid construct, contains the above vector or contains the above cell, and a pharmaceutically acceptable excipient.

[0031] Preferably, the pharmaceutical composition further contains an antitumor active drug.

[0032] Preferably, at least one of the above fusion protein, the above nucleic acid molecule, the above nucleic acid construct, the above vector, the above cell, the above pharmaceutical composition, and the above composition is used in the preparation of a drug for preventing, treating or adjuvant treating cancer, or delaying cancer progression, or reducing or inhibiting cancer recurrence in a subject.

[0033] In the present invention, the tumor preferably includes HER2-positive tumor cells, EGFR-positive tumor cells and PDL1-positive tumor cells. The EGFR-positive tumor preferably includes cervical cancer and breast cancer, such as EGFR + HeLa, EGFR + SKBR3 and EGFR + MDA-MB-231. The HER2-positive tumor preferably includes cervical cancer and breast cancer, such as HER2 + HeLa and HER2 + SKBR3. The PDL1-positive tumor preferably includes cervical cancer and breast cancer, such as PDL1 + HeLa and PDL1 + MDA-MB-231. The active ingredient in the drug preferably includes the above pro-tumor pyroptosis protein or its gene, the above immune pro-tumor pyroptosis protein targeting HER2, EGFR and PDL1 or its gene or biomaterial.

[0034] Further preferably, the tumor is breast cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, cholangiocarcinoma, gastric adenocarcinoma or glioblastoma.

[0035] Advantages of the present invention: The present invention proposes and designs a novel GSDMD fusion protein with controllable perforation activity outside cells and its application method. The fusion protein can specifically target the surface antigen of target cells outside cells and aggregate on the surface of target cells. On the basis of inhibiting the function of tumor cell surface antigens, further through the administration of the direct agonist DMB of GSDMD, the fusion protein can be controllably transformed into an active state, having the beneficial effect of inducing pyroptosis of target cells, and being able to effectively reduce the toxic and side effects brought by the administration of the small molecule drug DMB. In in vitro experiments, the fusion protein and the combined use method of the present invention have achieved good killing effects on a variety of tumor cell lines. Compared with the existing solutions, the solution proposed by the present invention can directly exert perforation activity outside cells, does not rely on endocytosis, does not rely on endogenous GSDMD and the cleavage of GSDMD protein, and is controllably activated to have perforation activity at a specific time and concentration by combining with an external direct agonist, which can be analogous to "switch-type" treatment. Therefore, it has better targeting and killing effects on target cells, improving clinical controllability and safety. Description of the drawings

[0036] Figure 1 It is a schematic diagram of the principle of the technical solution of the present invention.

[0037] Figure 2 It is a graph showing the purification results of the fusion protein GSDMD-EGFRn analyzed by molecular sieve chromatography and SDS-PAGE in Example 1.

[0038] Figure 3 It is a graph showing the purification results of the fusion protein GSDMD-PDL1.Nb analyzed by molecular sieve chromatography and SDS-PAGE in Example 2.

[0039] Figure 4 It is a graph showing the purification results of the fusion protein GSDMD-HER2.DARPin analyzed by molecular sieve chromatography and SDS-PAGE in Example 3.

[0040] Figure 5 It is a graph showing the results of detecting the binding ability of the fusion proteins GSDMD-EGFRn and GSDMD-PDL1.Nb to the surface antigen of HeLa cells by flow cytometry in Example 4.

[0041] Figure 6 It is a graph showing the results of detecting the binding ability of 3 fusion proteins to the surface antigen of SKBR3 cells by flow cytometry in Example 5.

[0042] Figure 7 Figure for the results of Example 6 using flow cytometry to detect the binding ability of three fusion proteins to the surface antigens of MDA-MB-231 cells.

[0043] Figure 8 Figure for Example 7 using fluorescence microscopy to evaluate the pyroptosis-promoting function of the present invention on EGFR-positive HeLa cells under different conditions; among them, Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0044] Figure 9 Statistical analysis of the pyroptosis-promoting function of the present invention on EGFR-positive HeLa cells; the scatter points therein represent the results of repeated experiments for each group of experiments; Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0045] Figure 10 Figure for Example 8 using fluorescence microscopy to evaluate the pyroptosis-promoting function of the present invention on PDL1-positive HeLa cells under different conditions; among them, Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0046] Figure 11 Statistical analysis of the pyroptosis-promoting function of the present invention on PDL1-positive HeLa cells; the scatter points therein represent the results of repeated experiments for each group of experiments; Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0047] Figure 12 Figure for Example 9 using fluorescence microscopy to evaluate the pyroptosis-promoting function of the present invention on HER2-positive SKBR3 cells under different conditions; among them, Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0048] Figure 13 Statistical analysis of the pyroptosis-promoting function of the present invention on HER2-positive SKBR3 cells; the scatter points therein represent the results of repeated experiments for each group of experiments; Hepes represents the buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control. Detailed implementation mode

[0049] The schematic diagram of the principle of the technical solution of the present invention is as follows Figure 1 As shown, in a specific implementation, the GSDMD fusion protein is a fusion protein obtained by linking GSDMD with ligands that recognize tumor cell surface antigens (such as EGFR, HER2, PDL1). The GSDMD fusion protein can achieve tumor cell membrane surface targeting and hindering effects extracellularly. Adding a GSDMD direct agonist (for example, the small molecule compound DMB) can relieve the auto-inhibited conformation of GSDMD in the fusion protein and activate its perforation function, thereby targeting the induction of pyroptosis in tumor cells, achieving the therapeutic effects of directly killing tumor cells and enhancing the anti-tumor immune response.

[0050] The following will describe the implementation of the present application in detail in conjunction with examples, but the present application is not limited to these examples. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents, etc. used are, unless otherwise specified, reagents and materials that can be obtained from commercial sources.

[0051] Example 1 Construction, Expression and Purification of GSDMD-EGFRn Fusion Protein (1) Design of the fusion protein: The fusion protein GSDMD-EGFRn consists of human GSDMD (fragment A) and EGFRn (the ligand of EGFR), and their amino acid sequences are shown in SEQ ID NO.1-2 respectively. The amino acid sequence of the Linker region is shown in SEQ ID NO.8, and the amino acid sequence of the finally obtained fusion protein GSDMD-EGFRn is shown in SEQ ID NO.5.

[0052] (2) Construction of the fusion protein plasmid: After codon optimization of the amino acid sequence of the GSDMD-EGFRn fusion protein in a prokaryotic expression system, the corresponding nucleotide sequence was obtained, as shown in SEQ ID NO.9. This sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. and cloned into the pET-28a(+)-SUMO vector using the 2X MultiF Seamless Assembly Mix kit from AB Clonal.

[0053] (3) Expression of the fusion protein: The recombinant vector was transformed into Escherichia coli Rosetta(DE3) competent cells, and positive clones were screened on a selective medium. A single colony was picked and inoculated into 3 mL of LB medium, supplemented with kanamycin (final concentration 50 μg / mL), and cultured overnight at 37°C with shaking at 220 rpm. Subsequently, 10 mL of the culture solution was inoculated into 500 mL of LB medium (containing 50 μg / mL kanamycin) and cultured at 37°C with shaking at 220 rpm until OD 600= 0.6 - 0.8. Subsequently, the culture temperature was reduced to 18 °C, IPTG (isopropyl-β-D-thiogalactoside, final concentration 0.3 mM) was added for induction of expression, and the culture was continued for 16 h. After the induction of expression was completed, the bacterial cell precipitate was collected by centrifugation at 4000 rpm for 30 min.

[0054] (4)Purification of the fusion protein: The collected bacterial cell precipitate was resuspended in pre-cooled 2×PBS buffer (containing 20 mM imidazole), and protease inhibitor PMSF (phenylmethylsulfonyl fluoride, final concentration 1 mM) was added. The resuspended bacterial cell suspension was disrupted by a high-pressure cell disruptor (flow rate 30 mL / min, pressure 700 bar, for 20 min). The disrupted bacterial cell suspension was centrifuged at 4 °C and 18000 rpm for 30 min, the precipitate was discarded, and the supernatant was collected. The fusion protein with His-SUMO tag was captured using Ni-NTA affinity chromatography packing material, and ULP1 enzyme was added to the protein elution solution to cleave the His-SUMO tag. Subsequently, the enzymatically digested protein was placed in a dialysis bag and dialyzed overnight at 4 °C. After dialysis was completed, the protein was purified again by Ni-NTA affinity chromatography column, the flow-through protein was collected, and concentrated by an ultrafiltration tube. The concentrated protein was dissolved in buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT), and further purified using a Superdex 200 Increase molecular sieve chromatography column (Cytiva), and the target protein fraction was collected.

[0055] The protein concentration was measured using a ultra-micro spectrophotometer, aliquoted, quickly frozen in liquid nitrogen, and stored at -80 °C. The purified protein was analyzed by reducing SDS-PAGE, and its electrophoresis pattern was as Figure 2 shown, showing a band with a molecular weight of approximately 61 kDa, which was consistent with the theoretical calculation (61.7 kDa), and the protein purity exceeded 90%.

[0056] Example 2 Construction, Expression and Purification of GSDMD-PDL1.Nb Fusion Protein

[0057] (1)Design of the fusion protein: The fusion protein GSDMD-PDL1.Nb consists of human GSDMD (fragment A) and a nanobody targeting PDL1, and their amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.3 respectively. The amino acid sequence of the Linker region is shown in SEQ ID NO.8, and the amino acid sequence of the finally obtained fusion protein GSDMD-PDL1.Nb is shown in SEQ IDNO.6.

[0058] (2)Construction of the fusion protein plasmid: After codon optimization of the amino acid sequence of the GSDMD-PDL1.Nb fusion protein in the prokaryotic expression system, the corresponding nucleotide sequence was obtained, as shown in SEQ ID NO.10. This sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. and cloned into the pET-28a(+)-SUMO vector using the 2X MultiF Seamless Assembly Mix kit from AB Clonal.

[0059] (3)The expression and purification steps of this fusion protein were the same as those of the GSDMD-EGFRn fusion protein in Example 1. The purified GSDMD-PDL1.Nb protein was analyzed by reducing SDS-PAGE, and its electrophoretogram was as Figure 3 shown, showing a band with a molecular weight of approximately 64 kDa, which was basically consistent with the theoretical calculation (68.0 kDa), and the protein purity exceeded 95%.

[0060] Example 3 Construction, Expression and Purification of GSDMD-HER2.DARPin Fusion Protein (1)Design of the fusion protein: The fusion protein GSDMD-HER2.DARPin consists of human-derived GSDMD (fragment A) and an antibody-like molecule engineered from the DARPin gene targeting HER2. Their amino acid sequences are shown in SEQ ID NO.1 and SEQ ID NO.4, respectively. The amino acid sequence of the Linker region is shown in SEQ ID NO.8, and the amino acid sequence of the finally obtained fusion protein GSDMD-HER2.DARPin is shown in SEQ ID NO.7.

[0061] (2)Construction of the fusion protein plasmid: After codon optimization of the amino acid sequence of the GSDMD-HER2.DARPin fusion protein in the prokaryotic expression system, the corresponding nucleotide sequence was obtained, as shown in SEQ ID NO.11. This sequence was synthesized by Shanghai Sangon Biotech Co., Ltd. and cloned into the pET-28a(+)-SUMO vector using the 2X MultiF Seamless Assembly Mix kit from AB Clonal.

[0062] (3)The expression and purification steps of this fusion protein were the same as those of the GSDMD-EGFRn fusion protein in Example 1. The purified GSDMD-HER2.DARPin protein was analyzed by reducing SDS-PAGE, and its electrophoretogram was as Figure 4 shown, showing a band with a molecular weight of approximately 66 kDa, which was basically consistent with the theoretical calculation (67.3 kDa), and the protein purity exceeded 90%.

[0063] Example 4 Detecting the binding level of the fusion protein to Hela cells by flow cytometry Adjust the density of EGFR + / PDL1 + Hela cells to 2×10 6 / mL. Take 200 μL of cells, wash them with pre-cooled PBS, and then incubate them with 200 μL of 5 μM, 1 μM, and 500 nM FITC-labeled GSDMD-EGFRn or GSDMD-PDL1.Nb fusion protein in PBS at 4°C in the dark for 1 h. After incubation, the cells are washed twice with cold PBS, then resuspended in 500 μL of PBS, and the change in the proportion of FITC-positive cell population is detected by flow cytometry. As Figure 5 The results show that the proportion of the fusion protein binding to Hela cells shows a concentration-dependent increasing trend and is much higher than that of the blank group, verifying the binding level of the fusion proteins GSDMD-EGFRn and GSDMD-PDL1.Nb to such cells and the cell surface antigen.

[0064] Example 5 Detecting the binding level of the fusion protein to SKBR3 cells by flow cytometry Adjust the density of EGFR + / PDL1 + / HER2 + SKBR3 cells to 2×10 6 / mL. Take 200 μL of cells, wash them with pre-cooled PBS, and then incubate them with 200 μL of 5 μM, 1 μM, and 500 nM FITC-labeled GSDMD-EGFRn, GSDMD-PDL1.Nb, or GSDMD-HER2.DARPin fusion protein in PBS at 4°C in the dark for 1 h. After incubation, the cells are washed twice with cold PBS, then resuspended in 500 μL of PBS, and the change in the proportion of FITC-positive cell population is detected by flow cytometry. As Figure 6 The results show that the proportion of the fusion protein binding to SKBR3 cells shows a concentration-dependent increasing trend and is much higher than that of the blank group, verifying the binding level of the three fusion proteins to such cells and the cell surface antigen.

[0065] Example 6 Detecting the binding level of the fusion protein to MDA-MB-231 cells by flow cytometry Adjust the density of EGFR + / PDL1 + / HER2 + MDA-MB-231 cells to 2×10 6 / mL, 200 μL of cells were taken. After being washed with pre-cooled PBS, they were incubated with 200 μL of 5 μM, 1 μM, and 500 nM FITC-labeled GSDMD-EGFRn, GSDMD-PDL1.Nb, or GSDMD-HER2.DARPin fusion proteins in PBS at 4°C in the dark for 1 h. After incubation, the cells were washed twice with cold PBS, then resuspended in 500 μL of PBS, and the proportion change of FITC-positive cell population was detected by flow cytometry. As Figure 7 The results showed that the proportion of the fusion proteins binding to MDA-MB-231 cells increased in a concentration-dependent manner and was much higher than that of the blank group, verifying the binding levels of the three fusion proteins to such cells and the cell surface antigens.

[0066] Example 7 Identification of the pyroptosis-promoting function of the present invention on EGFR-positive HeLa cells To verify the function of inducing pyroptosis in target cells by combining the fusion protein GSDMD-EGFRn with DMB, EGFR in the logarithmic growth phase + HeLa cells were digested and centrifuged with trypsin, and then cell counting and viability analysis were performed. The cells were seeded into 48-well plates, with 2.5×10 4 cells seeded in each well and cultured overnight so that the cell density did not exceed 50% when to be detected. After the cells were cultured overnight, different concentrations of the fusion protein GSDMD-EGFRn were incubated with the target cells for 1 h, and then different concentrations of the small molecule DMB (TargetMol, T36579) were added and incubated for 1 h. There were 3 replicates in each group of experiments. At the predetermined detection time point, a PI working solution (propidium iodide solution) at 40 μg / mL was prepared with pre-warmed serum-free cell culture medium. 100 μL of the dye working solution was added, and it was gently shaken to completely cover the cells, and incubated for 15 minutes. The dye working solution was aspirated, and the cells were washed 2 - 3 times with the culture medium, 5 minutes each time. The morphological changes of cell pyroptosis were observed and statistically analyzed using a fluorescence microscope. All data were statistically analyzed using GraphPad Prism 9.0. Unpaired t-tests were used for comparisons between two groups, and Two-way-ANOVA tests were used for comparisons among multiple groups. When p < 0.05, the differences between groups were considered statistically significant. Each group of experiments was repeated at least 3 times.

[0067] As Figure 8As shown, when full-length GSDMD protein is added alone outside tumor cells or the fusion protein GSDMD-EGFRn of the present invention is added alone without DMB activation, tumor cell membrane perforation cannot be induced, and pyroptosis cannot be induced, showing good safety. In sharp contrast, when the fusion protein GSDMD-EGFRn is used in combination with 5 μM direct agonist DMB, the test groups containing different concentrations of the fusion protein GSDMD-EGFRn and DMB significantly increased the number of pyroptotic cells compared with the test group that only added 5 μM DMB. At the same concentration, the synergistic effect of the GSDMD-EGFRn fusion protein and the direct agonist DMB is significantly greater than the effect of using the GSDMD-EGFRn fusion protein or the direct agonist DMB alone. As Figure 9 shown, the above results are statistically significant. The above results prove that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and greatly reduces the use concentration of the GSDMD-EGFRn fusion protein and the direct agonist DMB, which can reduce the toxic and side effects on non-target cells and achieve the purpose of safely and controllably directly killing the target tumor cells.

[0068] Example 8 Identification of the pyroptosis-promoting function of the present invention on PDL1-positive HeLa cells To verify the function of inducing pyroptosis of target cells after using the fusion protein GSDMD-PDL1.Nb in combination with DMB, PDL1 + HeLa cells in the logarithmic growth phase were digested and centrifuged with trypsin, and then cell counting and viability analysis were performed. The cells were seeded into a 48-well plate, with 2.5×10 4 cells seeded in each well and cultured overnight so that the cell density did not exceed 50% when to be detected. After the cells were cultured overnight, different concentrations of the fusion protein GSDMD-PDL1.Nb were incubated with the target cells for 1 h, and then different concentrations of the small molecule DMB were added and incubated for 1 h. There were 3 replicates in each group of experiments. After the predetermined detection time point, a PI working solution of 40 μg / mL was prepared with pre-warmed serum-free cell culture medium. 100 μL of the dye working solution was added, and it was gently shaken to completely cover the cells and incubated for 15 minutes. The dye working solution was aspirated, and the cells were washed 2-3 times with the culture medium, 5 minutes each time. The morphological changes of cell pyroptosis were observed and statistically analyzed using a fluorescence microscope. All data were statistically analyzed using GraphPad Prism 9.0. Unpaired t-tests were used for comparison between two groups, and Two-way-ANOVA tests were used for comparison among multiple groups. When p < 0.05, the differences between groups were considered statistically significant, and each group of experiments was repeated at least 3 times.

[0069] By Figure 10It can be seen that when DMB is not added for activation, adding the full-length GSDMD protein alone or the fusion protein GSDMD-PDL1.Nb of the present invention alone outside the tumor cells cannot cause tumor cell membrane perforation and cannot induce cell pyroptosis, showing good safety. In sharp contrast, when the fusion protein GSDMD-PDL1.Nb is used in combination with 5 μM direct agonist DMB, the test groups containing different concentrations of fusion protein GSDMD-PDL1.Nb and DMB significantly enhanced the number of cell pyroptosis compared with the test group to which only 5 μM DMB was added. At the same concentration, the synergistic effect of the GSDMD-PDL1.Nb fusion protein and the direct agonist DMB is significantly greater than the effect of using the GSDMD-PDL1.Nb fusion protein or the direct agonist DMB alone. Figure 11 As shown, the above results are statistically significant. The above results prove that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and greatly reduces the concentration of the GSDMD-PDL1.Nb fusion protein and the direct agonist DMB, which can reduce the toxic side effects on non-target cells and achieve the purpose of directly killing target tumor cells safely and controllably.

[0070] Example 9 Identification of the pyroptosis-promoting function of the present invention on HER2-positive SKBR3 cells In order to verify the function of inducing pyroptosis of target cells after the combination of the fusion protein and DMB, HER2 + SKBR3 cells were digested with trypsin and centrifuged, and then cell counting and activity analysis were performed. Cells were seeded into 48-well plates, with 4×10 cells per well. 4 The cells were cultured overnight so that the cell density did not exceed 50% when tested. After the cells were cultured overnight, the fusion protein GSDMD-HER2.DARPin with different concentrations was incubated with the target cells for 1 hour, and then different concentrations of small molecule DMB were added for incubation for 1 hour. Each group of experiments had 3 replicates. After the predetermined detection time point, a 40 μg / mL PI working solution was prepared with preheated serum-free cell culture medium. 100 μL of dye working solution was added, and the cells were completely covered with gentle shaking and incubated for 15 minutes. The dye working solution was aspirated, and the cells were washed with culture medium 2-3 times for 5 minutes each time. The morphological changes and statistical analysis of cell pyroptosis were observed using a fluorescence microscope. All data were statistically analyzed using GraphPad Prism 9.0. The comparison between the two groups was performed using an unpaired t test, and the comparison between multiple groups was performed using a Two-way-ANOVA test. When p < 0.05, the difference between the groups was considered statistically significant. Each group of experiments was repeated at least 3 times.

[0071] Depend on Figure 12It can be seen that when DMB activation is not added, adding the full-length GSDMD protein alone outside tumor cells or adding the fusion protein GSDMD-HER2.DARPin of the present invention alone cannot trigger tumor cell membrane perforation or induce pyroptosis, showing good safety. When the GSDMD-HER2.DARPin fusion protein is used in combination with a low concentration (2.5 μM) of the direct agonist DMB, the test groups containing different concentrations of the fusion protein GSDMD-HER2.DARPin and DMB significantly enhanced the proportion of pyroptosis compared with the test group only adding 2.5 μM DMB. At the same concentration, the synergistic effect of the GSDMD-HER2.DARPin fusion protein and the direct agonist DMB is significantly greater than the effect of using the GSDMD-HER2.DARPin fusion protein or the direct agonist DMB alone. As Figure 13 shown, the above results are statistically significant. The above results prove that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and greatly reduces the use concentrations of the GSDMD-HER2.DARPin fusion protein and the direct agonist DMB, which can reduce the toxic and side effects on non-target cells and achieve the purpose of safely and controllably directly killing target tumor cells.

[0072] The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A GSDMD fusion protein having controllable perforation activity outside a cell, characterized in that: The GSDMD fusion protein comprises fragment A and fragment B, The fragment A is a polypeptide fragment at position XY of the human GSDMD protein sequence, which is in an autoinhibitory state, wherein X is selected from an integer between 1 and 6, and Y is selected from an integer between 479 and 484. The amino acid sequence of the human GSDMD protein is shown in SEQ ID No. 1; The fragment B is a polypeptide fragment that targets and recognizes a cell surface antigen, and the cell surface antigen is EGFR, PDL1 or HER2. The amino acid sequence of the fragment B is shown in SEQ ID No. 2-4.

2. The GSDMD fusion protein according to claim 1, characterized in that The fragment A is directly or indirectly connected to the N-terminus or C-terminus of the fragment B, and a plurality of the fragments A are directly or indirectly connected to the N-terminus or C-terminus of a plurality of the fragments B.

3. The GSDMD fusion protein according to claim 1, characterized in that The fusion protein sequence is shown in SEQ ID NO.5-7.

4. A composition of a GSDMD fusion protein and a GSDMD direct agonist, characterized in that: The composition is a combination of any GSDMD fusion protein according to claims 1 to 3 and a GSDMD direct agonist, wherein the GSDMD direct agonist can directly activate the perforation activity of the GSDMD protein without cleaving the GSDMD protein.

5. The composition according to claim 4, characterized in that The GSDMD direct agonist is a small molecule compound 6,7-dichloro-2-methanesulfonyl-3-N-tert-butylaminoquinoxaline, or a 6,7-dichloro-2-methanesulfonyl-3-N-tert-butylaminoquinoxaline derivative having the function of directly activating GSDMD.

6. A biomaterial related to the GSDMD fusion protein according to any one of claims 1 to 3, characterized in that: The biological material is any one of the following: (1) A nucleic acid molecule encoding the GSDMD fusion protein according to any one of claims 1 to 3; (2) an expression cassette containing the nucleic acid molecule described in (1); (3) a recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2); (4) a recombinant microorganism containing the nucleic acid molecule described in (1), or a recombinant microorganism containing the expression cassette described in (2), or a recombinant microorganism containing the recombinant vector described in (3); (5) A cell line containing the nucleic acid molecule described in (1), or a cell line containing the expression cassette described in (2), or a cell line containing the recombinant vector described in (3).

7. Use of the GSDMD fusion protein according to claims 1 to 3, the composition according to claim 4 or 5, or the biomaterial according to claim 6 in the preparation of a drug that directly kills target cells.

8. The use according to claim 7, characterized in that: When used, the GSDMD fusion protein is expressed, and the GSDMD fusion protein recognizes the target cell surface antigen and aggregates on the extracellular surface. The GSDMD direct agonist that does not cleave the GSDMD protein is applied to convert the GSDMD fusion protein on the extracellular surface into a perforating active state, which is independent of cell endocytosis and protease cleavage, forms holes on the tumor cell membrane and induces cell pyroptosis, thereby directly killing the target cells.

9. Use of the GSDMD fusion protein according to any one of claims 1 to 3, the composition according to claim 4 or 5, or the biomaterial according to claim 6 in the preparation of anti-tumor drugs.

10. The use according to claim 8 or 9, characterized in that: The tumor type is selected from EGFR, PDL1 or HER2 positive tumor cells.

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