A GSDMD fusion protein with controllable perforation activity outside cells and its application

Abstract

The present invention discloses a GSDMD fusion protein with controllable perforation activity outside cells and its application. The present invention connects a GSDMD protein in a self-inhibited state with a polypeptide fragment that targets and recognizes tissues or cells. The formed fusion protein can specifically bind to and aggregate on the surface of target cells outside cells. Through the use of a GSDMD direct agonist, the GSDMD protein contained in the fusion protein is converted from an inactive state to an active state without being cleaved, and then forms pores in the cell membrane of tumor cells to induce cell pyroptosis, and can effectively reduce the toxic side effects caused by the use of the small molecule drug DMB. In in vitro experiments, the composition and use method of the present invention achieved good killing effects on various tumor cell lines. The present invention does not rely on cell endocytosis, does not rely on the cleavage of endogenous GSDMD protein and protease, and therefore has better targeting and killing effects on target cells.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a GSDMD fusion protein with controllable perforation activity outside cells and applications thereof. Background Art

[0002] Pyroptosis is a type of programmed cell death triggered by members of the gasdermin family of proteins, including GSDMD, which are localized in the cytoplasm. GSDMD consists of two conserved domains: the N-terminal domain (GSDMD-N) and the C-terminal domain (GSDMD-C). GSDMD-N is the primary functional domain, while GSDMD-C has an autoinhibitory effect. When GSDMD is cleaved by inflammatory caspases, the released GSDMD-N domain forms pores on the inner surface of the cell membrane, leading to cell swelling, membrane perforation, and the release of cellular contents, thereby activating a strong inflammatory and immune response.

[0003] Existing research (patent publication number CN118063584A) has discovered a GSDMD variant created by genetically engineering a cathepsin D cleavage site between the N-terminal and C-terminal domains of GSDMD. This GSDMD variant is then linked to an antibody to form a fusion protein. This fusion protein is targeted to specific cell surfaces and then internalized. Upon cleavage by cathepsin D within the cell, the GSDMD-N-terminal domain is released, forming pores in the cell membrane and inducing pyroptosis. The main drawback of this approach is that the fusion protein cannot exert its permeation function outside the cell and must rely on endocytosis and intracellular protease cleavage to cleave the GSDMD-N-terminal domain. Furthermore, the activation process and timing of cell membrane permeation cannot be precisely controlled.

[0004] Existing research has also found (DOI: 10.1016 / j.heliyon.2024.e30444.) that a fusion protein is constructed by combining the HER2 antibody P1h3, an albumin-binding peptide, a cleavage site for cathepsin B, an endosomal-disrupting peptide E5C3, and the GSDMD-N-terminal domain. This fusion protein specifically recognizes HER2-positive tumor cells and is internalized. Once inside the endosome, it is cleaved by cathepsin B, releasing the GSDMD-N-terminal domain, which enters the cytoplasm during E5C3-mediated endosomal escape, ultimately inducing pyroptosis in tumor cells. However, a major limitation of this strategy is that the fusion protein cannot exert its membrane permeation function outside the cell; it still relies on endocytosis and endogenous cleavage to cleave the GSDMD-N-terminal domain within the cell. Furthermore, the activation process and timing of membrane permeation cannot be precisely controlled. Furthermore, due to its complex design, the activity of this fusion protein is affected by numerous factors.

[0005] Existing research has also found (DOI: 10.1016 / j.cell.2024.08.007.) that the small molecule GSDMD agonist DMB (6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline) can directly activate endogenous GSDMD without cleaving it, causing pores on the inner surface of the cell membrane and triggering pyroptosis. This approach has major limitations: GSDMD does not naturally exist outside cells, DMB's agonistic effects are limited to intracellular GSDMD, and it cannot be targeted to 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 in the fight against cancer and is a promising therapeutic strategy with great research significance. However, current research on related drugs and therapies is still in its early stages. Summary of the Invention

[0007] Existing GSDMD fusion protein technology is generally limited to the following: it cannot exert membrane perforation effects outside the cell, relies on endocytosis and protease cleavage, and requires the GSDMD-N-terminal domain to be released inside the target cell to trigger cell membrane perforation and promote cell pyroptosis. The present invention addresses the deficiencies in the existing technology and provides a GSDMD fusion protein and application scheme with controllable perforation activity outside the cell. The GSDMD fusion protein and application scheme designed by the present invention realize the cell membrane perforation function of GSDMD outside the cell, and do not rely on endocytosis, do not need to be cleaved by proteases to release the GSDMD-N-terminal domain, and through combination with external direct agonists, the perforation activity is controllably activated at a specific time and concentration, which can be compared to "switch-type" treatment, improve clinical controllability and safety, and achieve precise killing of tumor cells and immune activation, which is significantly better than the limitations of existing technologies.

[0008] In order to achieve the above-mentioned purpose, the specific technical solutions of the present invention are as follows:

[0009] The present invention provides a GSDMD fusion protein with controllable perforation activity outside cells. Specifically, a human GSDMD protein polypeptide fragment (fragment A) is linked to a polypeptide fragment (fragment B) that targets an antigen on the extracellular surface of tumor cells to form a novel fusion protein, wherein:

[0010] Fragment A is a polypeptide fragment at position XY of the human GSDMD protein sequence, which is naturally in an autoinhibited state, wherein X is selected from an integer between 1 and 6, and Y is selected from an integer between 479 and 484, and comprises the N-terminal domain and the C-terminal domain of the GSDMD protein;

[0011] Fragment B is a polypeptide fragment that targets and recognizes tissues or cells.

[0012] The fusion protein described in the present invention may include one or more fragments A and one or more fragments B, and the present invention does not limit their number of repetitions and connection order. When the number of fragment repetitions is not 1, the sequences of the 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. In order to maintain the integrity of the structure of fragments A and fragment B, the fusion protein may or may not include a linker, and the present invention does not limit this. In the present invention, the linker is located between two adjacent fragments, for example, between fragment A and fragment B, or between two fragments A, or between two fragments B, and the present invention does not limit this.

[0013] The present invention also provides a method for combining the GSDMD fusion protein with a GSDMD direct agonist ( Figure 1 ). The GSDMD direct agonist can directly activate the perforation activity of GSDMD without cleaving GSDMD. Specifically: expressing and preparing the GSDMD fusion protein of the present invention. The GSDMD fusion protein can specifically target the target cell surface antigen outside the cell and aggregate to the outer surface of the target cell. In this state, the fusion protein only exerts a targeted inhibitory function on the tumor cell outer surface antigen through fragment B, but does not have the activity of cell membrane perforation, thereby reducing the toxic side effects on the cells. At a specified time, a specified concentration of GSDMD direct agonist is applied, which can controllably convert fragment A of the GSDMD fusion protein into a membrane perforation active state without being cleaved, thereby forming pores on the outer surface of the tumor cell membrane and inducing pyroptosis of the target cell, achieving the therapeutic effect of directly killing tumor cells and enhancing the anti-tumor immune response.

[0014] The present invention discovered that adding either the full-length GSDMD protein or the GSDMD fusion protein of the present invention alone to tumor cells failed to induce tumor cell membrane perforation. Furthermore, adding the full-length GSDMD protein in combination with the small molecule agonist DMB to tumor cells also failed to induce perforation. In stark contrast, the combined use of the GSDMD fusion protein of the present invention and the direct agonist DMB effectively perforated the tumor cell outer membrane and induced pyroptosis. Furthermore, at the same concentration, the synergistic effect of the GSDMD fusion protein and the direct agonist DMB was significantly greater than that of either the GSDMD fusion protein or the direct agonist DMB alone. This significantly reduces the concentration of the GSDMD fusion protein and the direct agonist, minimizing toxic side effects on non-target cells and achieving safe and controllable direct killing of target tumor cells. The present invention is simple and efficient, independent of cellular endocytosis and cleavage by intracellular proteases, significantly reducing factors limiting its activity. Furthermore, the present invention can be combined with other tumor treatments to enhance anti-tumor therapeutic efficacy.

[0015] Preferably, the amino acid sequence of the human GSDMD protein is as shown in SEQ ID No. 1, or the sequence shown in a variant thereof.

[0016] Preferably, the polypeptide fragment at positions 6-479 of the human GSDMD protein sequence, or the sequence shown by a variant thereof is selected.

[0017] Preferably, fragment B of the fusion protein is a polypeptide fragment targeting the tumor cell surface antigen EGFR, PDL1 or HER2, and the polypeptide fragment is derived from EGFRn, nanoantibodies targeting PDL1 and genetically engineered antibodies DARPin targeting HER2 reported in the literature, and the amino acid sequence is shown in SEQ ID No. 2-4, or the sequence shown in its variant.

[0018] In many embodiments, fragment B binds to tumor cell surface antigens 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.

[0019] 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 the sequence shown in a variant thereof.

[0020] Preferably, the amino acid sequence of the fusion protein is as shown in SEQ ID No. 5-7, or a sequence shown in a variant thereof.

[0021] wherein 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.

[0022] The present invention also provides a composition comprising any of the aforementioned GSDMD fusion proteins and a GSDMD direct agonist, wherein the GSDMD direct agonist can directly activate the perforation activity of GSDMD without cleaving GSDMD.

[0023] Preferably, the GSDMD direct agonist is a small molecule compound 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline (DMB), or a DMB derivative that has the ability to directly activate GSDMD. In various embodiments of the present invention, the effective concentration of DMB is less than or equal to 5 μM, which is significantly lower than its concentration for endogenous GSDMD agonist activity (EC 50 =5~10 μM), thereby effectively avoiding the impact of DMB on non-target cells and reducing cytotoxicity.

[0024] The present invention also provides a biomaterial related to the above-mentioned GSDMD fusion protein, wherein the biomaterial is any one of the following:

[0025] (1) a nucleic acid molecule encoding the aforementioned GSDMD fusion protein;

[0026] (2) an expression cassette containing the nucleic acid molecule described in (1);

[0027] (3) a recombinant vector containing the nucleic acid molecule described in (1), or a recombinant vector containing the expression cassette described in (2);

[0028] (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);

[0029] (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).

[0030] Specifically:

[0031] Preferably, a nucleic acid molecule can encode the above-mentioned fusion protein.

[0032] Preferably, a nucleic acid construct comprises the above nucleic acid molecule.

[0033] 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.

[0034] Preferably, a cell secretes the above-mentioned fusion protein, contains the above-mentioned nucleic acid molecule, contains the above-mentioned nucleic acid construct or contains the above-mentioned expression vector.

[0035] In one embodiment, the cells include immune cells.

[0036] In one embodiment, the cells include T cells.

[0037] In one embodiment, the cell comprises a genetically engineered cell.

[0038] Preferably, a kit comprising the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct or the above-mentioned expression vector is used to prepare the above-mentioned fusion protein or the above-mentioned cell.

[0039] Preferably, a pharmaceutical composition comprises the aforementioned nucleic acid molecule, the aforementioned nucleic acid construct, the aforementioned vector or the aforementioned cell, and pharmaceutically acceptable excipients.

[0040] Preferably, the pharmaceutical composition further comprises an anti-tumor drug.

[0041] Preferably, at least one of the above-mentioned fusion protein, the above-mentioned nucleic acid molecule, the above-mentioned nucleic acid construct, the above-mentioned vector, the above-mentioned cell, the above-mentioned pharmaceutical composition, and the above-mentioned composition is used in the preparation of a drug for preventing, treating, or assisting in the treatment of cancer, or delaying cancer progression, or reducing or inhibiting cancer recurrence in a subject.

[0042] 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 + The HER2 positive tumors preferably include cervical cancer and breast cancer, such as HER2 + HeLa and HER2 + SKBR3. The PDL1-positive tumors preferably include cervical cancer and breast cancer, such as PDL1 + HeLa and PDL1 + The active ingredients in the drug preferably include the above-mentioned tumor-promoting pyroptosis protein or its gene, the above-mentioned immune tumor-promoting pyroptosis protein targeting HER2, EGFR and PDL1 or its gene or biological material.

[0043] Further preferably, the tumor is breast cancer, colon cancer, ovarian cancer, prostate cancer, lung cancer, pancreatic cancer, renal cell carcinoma, cervical cancer, endometrial cancer, bile duct cancer, gastric adenocarcinoma or glioblastoma.

[0044] Beneficial effects of the present invention:

[0045] This invention proposes and designs a novel GSDMD fusion protein with controllable extracellular perforation activity and its application method. This fusion protein can specifically target target cell surface antigens and aggregate on the target cell surface. In addition to inhibiting tumor cell surface antigen function, administration of the GSDMD direct agonist DMB allows the fusion protein to be controllably switched to an active state, beneficially inducing pyroptosis in target cells while effectively reducing the toxic side effects associated with the small molecule drug DMB. In vitro experiments show that the fusion protein and combined use method of the present invention have demonstrated good anti-tumor activity against various tumor cell lines. Compared to existing approaches, the proposed approach can directly exert its perforation activity extracellularly, independent of endocytosis, endogenous GSDMD, or GSDMD protein cleavage. By combining it with an external direct agonist, the perforation activity can be controllably activated at a specific time and concentration, analogous to an "on-off" therapy. This provides better targeting and anti-tumor activity against target cells, improving clinical controllability and safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is a schematic diagram of the principle of the technical solution of the present invention.

[0047] Figure 2 This is a diagram showing the purification results of the fusion protein GSDMD-EGFRn analyzed by molecular sieve chromatography and SDS-PAGE in Example 1.

[0048] Figure 3 This is a diagram showing the purification results of the fusion protein GSDMD-PDL1.Nb analyzed by molecular sieve chromatography and SDS-PAGE in Example 2.

[0049] Figure 4 This is a diagram showing the purification results of the fusion protein GSDMD-HER2.DARPin analyzed by molecular sieve chromatography and SDS-PAGE in Example 3.

[0050] Figure 5 This is a graph showing the results of Example 4 using flow cytometry to detect the binding ability of the fusion proteins GSDMD-EGFRn and GSDMD-PDL1.Nb to HeLa cell surface antigens.

[0051] Figure 6This is a graph showing the results of Example 5 using flow cytometry to detect the binding ability of three fusion proteins to SKBR3 cell surface antigens.

[0052] Figure 7 This is a graph showing the results of Example 6 using flow cytometry to detect the binding ability of three fusion proteins to MDA-MB-231 cell surface antigens.

[0053] Figure 8 In Example 7, fluorescence microscopy was used to evaluate the pyroptosis-promoting effect of the present invention on EGFR-positive HeLa cells under different conditions; wherein, Hepes represents a buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0054] Figure 9 This is a statistical analysis of the pyroptosis-promoting effect of the present invention on EGFR-positive HeLa cells; the scattered points represent the repeated experimental results of each group of experiments; Hepes represents buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0055] Figure 10 In Example 8, fluorescence microscopy was used to evaluate the pyroptosis-promoting effect of the present invention on PDL1-positive HeLa cells under different conditions; wherein, Hepes represents a buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0056] Figure 11 Figure 2 is a statistical analysis of the pyroptosis-promoting effect of the present invention on PDL1-positive HeLa cells; the scattered points represent the repeated experimental results of each group of experiments; Hepes represents buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0057] Figure 12 In Example 9, fluorescence microscopy was used to evaluate the pyroptosis-promoting effect of the present invention on HER2-positive SKBR3 cells under different conditions; wherein, Hepes represents a buffer solution (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control.

[0058] Figure 13 This is a statistical analysis of the pyroptosis-promoting effect of the present invention on HER2-positive SKBR3 cells; the scattered points represent the repeated experimental results of each group of experiments; Hepes represents buffer (20 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM DTT) as a blank control. DETAILED DESCRIPTION

[0059] The schematic diagram of the technical solution principle of the present invention is as follows Figure 1 As shown, in a specific embodiment, the GSDMD fusion protein is a fusion protein in which GSDMD is linked to a ligand that recognizes tumor cell surface antigens (such as EGFR, HER2, and PDL1). The GSDMD fusion protein can achieve targeting and blocking effects on the tumor cell membrane surface outside the cell. Adding a GSDMD direct agonist (for example, the small molecule compound DMB) can release the autoinhibitory conformation of GSDMD in the fusion protein and activate its perforation function, thereby targetedly inducing pyroptosis in tumor cells, achieving the therapeutic effect of directly killing tumor cells and enhancing anti-tumor immune responses.

[0060] The embodiments of the present application will be described in detail below with reference to the examples, but the present application is not limited to these examples. The experimental methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0061] Example 1 Construction, expression and purification of GSDMD-EGFRn fusion protein

[0062] (1) Design of fusion protein: The fusion protein GSDMD-EGFRn is composed of human GSDMD (fragment A) and EGFRn (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 final fusion protein GSDMD-EGFRn is shown in SEQ ID NO. 5.

[0063] (2) Construction of fusion protein plasmid: The amino acid sequence of the GSDMD-EGFRn fusion protein was codon-optimized in the prokaryotic expression system to obtain the corresponding nucleotide sequence, 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.

[0064] (3) Expression of fusion protein: The recombinant vector was transformed into Escherichia coli Rosetta (DE3) competent cells, and positive clones were screened on selective medium. Single clones were picked and inoculated into 3 mL LB medium, kanamycin (final concentration 50 μg / mL) was added, and cultured at 37°C, 220 rpm, and shaken overnight. Subsequently, 10 mL of culture solution was inoculated into 500 mL LB medium (containing 50 μg / mL kanamycin), and cultured at 37°C, 220 rpm, and shaken until OD 600 =0.6-0.8. The culture temperature was then lowered to 18°C, and IPTG (isopropyl-β-D-thiogalactopyranoside, final concentration 0.3 mM) was added to induce expression. Culture was continued for 16 hours. After induction, the pellet was collected by centrifugation at 4000 rpm for 30 minutes.

[0065] (4) Purification of fusion protein: The collected bacterial pellet was resuspended in pre-cooled 2× PBS buffer (containing 20 mM imidazole) and the protease inhibitor PMSF (phenylmethylsulfonyl fluoride, final concentration 1 mM) was added. The resuspended bacterial solution was disrupted by a high-pressure cell disruptor (flow rate 30 mL / min, pressure 700 bar, duration 20 min). The disrupted bacterial solution was centrifuged at 4°C and 18,000 rpm for 30 min, the precipitate was discarded, and the supernatant was collected. The fusion protein with the His-SUMO tag was captured using Ni-NTA affinity chromatography filler, and ULP1 enzyme was added to the protein eluate to cleave the His-SUMO tag. Subsequently, the enzymatically digested protein was placed in a dialysis bag and dialyzed at 4°C overnight. After dialysis, the protein was purified again by Ni-NTA affinity chromatography column, the flow-through protein was collected, and concentrated by 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) to collect the target protein fraction.

[0066] The protein concentration was determined using an ultra-micro spectrophotometer, and the aliquots were quickly frozen in liquid nitrogen and stored at -80°C. The purified protein was analyzed by reducing SDS-PAGE, and the electrophoresis pattern was as follows: Figure 2 As shown, a band with a molecular weight of approximately 61 kDa was displayed, which was consistent with the theoretical calculation (61.7 kDa), and the protein purity was over 90%.

[0067] Example 2 Construction, expression and purification of GSDMD-PDL1.Nb fusion protein

[0068] (1) Design of fusion protein: The fusion protein GSDMD-PDL1.Nb consists of human GSDMD (fragment A) and a nanobody targeting PDL1. The 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. The amino acid sequence of the resulting fusion protein GSDMD-PDL1.Nb is shown in SEQ ID NO.6.

[0069] (2) Construction of fusion protein plasmid: The amino acid sequence of the GSDMD-PDL1.Nb fusion protein was codon-optimized in the prokaryotic expression system to obtain the corresponding nucleotide sequence, 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.

[0070] (3) The expression and purification steps of the fusion protein are consistent with those of the GSDMD-EGFRn fusion protein in Example 1. The purified GSDMD-PDL1.Nb protein was analyzed by reducing SDS-PAGE, and its electrophoretic pattern was as follows: Figure 3 As shown, a band with a molecular weight of approximately 64 kDa was displayed, which was basically consistent with the theoretical calculation (68.0 kDa), and the protein purity was over 95%.

[0071] Example 3 Construction, expression and purification of GSDMD-HER2.DARPin fusion protein

[0072] (1) Design of fusion protein: The fusion protein GSDMD-HER2.DARPin consists of human GSDMD (fragment A) and a genetically engineered antibody-like protein targeting HER2, the amino acid sequences of which 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 resulting fusion protein GSDMD-HER2.DARPin is shown in SEQ ID NO.7.

[0073] (2) Construction of fusion protein plasmid: The amino acid sequence of the GSDMD-HER2.DARPin fusion protein was codon-optimized in the prokaryotic expression system to obtain the corresponding nucleotide sequence, 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.

[0074] (3) The expression and purification steps of the fusion protein are consistent with those of the GSDMD-EGFRn fusion protein in Example 1. The purified GSDMD-HER2.DARPin protein was analyzed by reducing SDS-PAGE, and its electrophoretic pattern was as follows: Figure 4 As shown, a band with a molecular weight of approximately 66 kDa was displayed, which was basically consistent with the theoretical calculation (67.3 kDa), and the protein purity was over 90%.

[0075] Example 4 Flow cytometry was used to detect the binding level of the fusion protein to Hela cells

[0076] EGFR + / PDL1 + The HeLa cell density was adjusted to 2 × 10 6 / mL, 200 μL of cells were taken, washed with pre-chilled PBS, and then incubated with 200 μL of 5 μM, 1 μM, and 500 nM FITC-labeled GSDMD-EGFRn or GSDMD-PDL1.Nb fusion proteins in PBS at 4°C in the dark for 1 h. After incubation, the cells were washed twice with cold PBS and then resuspended in 500 μL PBS. The changes in the proportion of FITC-positive cell populations were detected by flow cytometry. Figure 5 The results showed that the ratio of fusion protein binding to Hela cells increased in a concentration-dependent manner and was 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 antigens.

[0077] Example 5 Detection of the binding level of the fusion protein to SKBR3 cells using flow cytometry

[0078] EGFR + / PDL1 + / HER2 + The density of SKBR3 cells was adjusted to 2 × 10 6 / mL, 200 μL of cells were taken, washed with pre-chilled PBS, and then 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 and then resuspended in 500 μL PBS. The changes in the proportion of FITC-positive cell populations were detected by flow cytometry. Figure 6The results showed that the ratio of fusion proteins binding to SKBR3 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.

[0079] Example 6 Flow cytometry was used to detect the binding level of the fusion protein to MDA-MB-231 cells

[0080] EGFR + / PDL1 + / HER2 + The density of MDA-MB-231 cells was adjusted to 2 × 10 6 / mL, 200 μL of cells were taken, washed with pre-chilled PBS, and then 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 and then resuspended in 500 μL PBS. The changes in the proportion of FITC-positive cell populations were detected by flow cytometry. Figure 7 The results showed that the ratio of 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.

[0081] Example 7 Identification of the Pyroptosis-Promoting Function of the Present Invention on EGFR-Positive HeLa Cells

[0082] In order to verify the function of inducing pyroptosis of target cells after the fusion protein GSDMD-EGFRn is used in combination with DMB, EGFR in the logarithmic growth phase is + HeLa cells were digested with trypsin and centrifuged, and then cell count and activity analysis were performed. Cells were seeded into 48-well plates at a rate of 2.5 × 10 cells per well. 4Cells were cultured overnight, ensuring that the cell density did not exceed 50% at the time of assay. After overnight incubation, target cells were incubated with various concentrations of the fusion protein GSDMD-EGFRn for 1 hour. Different concentrations of the small molecule DMB (Shanghai Taoshu, T36579) were then added for 1 hour. Each experiment was performed in triplicate. At the scheduled assay time point, a 40 μg / mL PI working solution (propidium iodide solution) was prepared in prewarmed serum-free cell culture medium. 100 μL of the dye working solution was added, 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 culture medium for 5 minutes each. Morphological changes in pyroptosis were observed using a fluorescence microscope and statistical analysis was performed. All data were analyzed using GraphPad Prism 9.0. Unpaired t-tests were used for comparisons between two groups, and two-way ANOVA was used for comparisons between multiple groups. Differences between groups were considered statistically significant when p < 0.05. Each experiment was repeated at least three times.

[0083] like Figure 8 As shown, when DMB is not added for activation, the addition of full-length GSDMD protein alone or the fusion protein GSDMD-EGFRn of the present invention alone outside the tumor cells cannot trigger tumor cell membrane perforation and cannot induce cell pyroptosis, 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 fusion protein GSDMD-EGFRn 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-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 As shown, the above results are statistically significant. The above results demonstrate that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and significantly reduces the concentration of the GSDMD-EGFRn fusion protein and the direct agonist DMB, which can reduce toxic side effects on non-target cells and achieve the purpose of directly killing target tumor cells safely and controllably.

[0084] Example 8 Identification of the Pyroptosis-Promoting Function of the Present Invention on PDL1-Positive HeLa Cells

[0085] In order to verify the function of inducing pyroptosis of target cells by combining the fusion protein GSDMD-PDL1.Nb with DMB, PDL1 in the logarithmic growth phase was cultured. +HeLa cells were digested with trypsin and centrifuged, and then cell count and activity analysis were performed. Cells were seeded into 48-well plates at a rate of 2.5 × 10 cells per well. 4 Cells were cultured overnight, ensuring that the cell density did not exceed 50% at the time of assay. After overnight incubation, target cells were incubated with various concentrations of the fusion protein GSDMD-PDL1.Nb for 1 hour, followed by incubation with various concentrations of the small molecule DMB for 1 hour. Each experiment was performed in triplicate. At the scheduled assay time point, a 40 μg / mL PI working solution was prepared in prewarmed serum-free cell culture medium. 100 μL of the dye working solution was added, gently agitated 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 culture medium for 5 minutes each. Morphological changes in pyroptosis were observed using a fluorescence microscope and statistical analysis was performed. All data were analyzed using GraphPad Prism 9.0. Unpaired t-tests were used for comparisons between two groups, and two-way ANOVA was used for comparisons between multiple groups. Differences between groups were considered statistically significant when p < 0.05. Each experiment was repeated at least three times.

[0086] Depend on Figure 10 It 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 to the outside of 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. As Figure 11 As shown, the above results are statistically significant. The above results demonstrate that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and significantly reduces the concentration of the GSDMD-PDL1.Nb fusion protein and the direct agonist DMB, which can reduce toxic side effects on non-target cells and achieve the purpose of directly killing target tumor cells safely and controllably.

[0087] Example 9 Identification of the Pyroptosis-Promoting Function of the Present Invention on HER2-Positive SKBR3 Cells

[0088] To verify the function of inducing pyroptosis in target cells after the fusion protein is used in combination with DMB, HER2+ SKBR3 cells were digested with trypsin and centrifuged, and then cell count and activity analysis were performed. Cells were seeded into 48-well plates at 4×10 cells per well. 4 Cells were cultured overnight, ensuring that the cell density did not exceed 50% at the time of assay. After overnight incubation, target cells were incubated with various concentrations of the fusion protein GSDMD-HER2.DARPin for 1 hour, followed by incubation with various concentrations of the small molecule DMB for 1 hour. Each experiment was performed in triplicate. At the scheduled assay time point, a 40 μg / mL PI working solution was prepared in prewarmed serum-free cell culture medium. 100 μL of the dye working solution was added, gently agitated 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 culture medium for 5 minutes each. Morphological changes in pyroptosis were observed using a fluorescence microscope and statistical analysis was performed. All data were analyzed using GraphPad Prism 9.0. Unpaired t-tests were used for comparisons between two groups, and two-way ANOVA was used for comparisons between multiple groups. Differences between groups were considered statistically significant when p < 0.05. Each experiment was repeated at least three times.

[0089] Depend on Figure 12 It can be seen that when DMB is not added for activation, adding the full-length GSDMD protein alone or the fusion protein GSDMD-HER2.DARPin of the present invention alone to the outside of the tumor cells cannot cause tumor cell membrane perforation and cannot induce cell 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 group containing different concentrations of the fusion protein GSDMD-HER2.DARPin and DMB significantly enhanced the proportion of cell pyroptosis compared with the test group to which only 2.5 μM DMB was added. 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. Figure 13 As shown, the above results are statistically significant. The above results demonstrate that the present invention has the effect of inducing pyroptosis of target cells under controllable conditions, and significantly reduces the concentration of the GSDMD-HER2.DARPin fusion protein and the direct agonist DMB, which can reduce toxic side effects on non-target cells and achieve the purpose of directly killing target tumor cells safely and controllably.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A GSDMD fusion protein with controllable perforation activity outside the 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 autoinhibited 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, wherein the cell surface antigen is EGFR, PDL1 or HER2, and the amino acid sequence of the fragment B is shown in SEQ ID No. 2-4; The fusion protein sequence is shown in SEQ ID NO.5-7.

2. 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 claim 1 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; The GSDMD direct agonist is a small molecule compound 6,7-dichloro-2-methanesulfonyl-3-N-tert-butylaminoquinoxaline.

3. The biomaterial related to the GSDMD fusion protein according to claim 1, characterized in that The biological material is any one of the following: (1) A nucleic acid molecule encoding the GSDMD fusion protein of claim 1; (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).

4. Use of the GSDMD fusion protein of claim 1, the composition of claim 2, or the biomaterial of claim 3 in the preparation of a drug for causing pyroptosis in tumor cells, wherein the tumor cells are selected from EGFR-, PDL1-, or HER2-positive cervical cancer or breast cancer cells.

5. The use according to claim 4, characterized in that During application, the GSDMD fusion protein is expressed, and the GSDMD fusion protein recognizes the target cell surface antigen and accumulates on the extracellular surface. The small molecule compound 6,7-dichloro-2-methylsulfonyl-3-N-tert-butylaminoquinoxaline, which does not cleave the GSDMD protein, is applied to convert the GSDMD fusion protein on the extracellular surface into a perforation active state, which forms pores on the tumor cell membrane and triggers cell pyroptosis independently of cell endocytosis and protease cleavage, thereby directly killing the target cells.

6. Use of the GSDMD fusion protein according to claim 1, the composition according to claim 2, or the biomaterial according to claim 3 in the preparation of an anti-tumor drug; The tumor type is selected from EGFR, PDL1 or HER2 positive cervical cancer or breast cancer cells.

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