Application of disulfide cyclopeptide and biomaterial containing disulfide cyclopeptide in tumor resistance

Abstract

The invention discloses application of a disulfide cyclopeptide and a biomaterial containing the disulfide cyclopeptide in tumor resistance, and the biomaterial comprises a bacterial or viral vector containing a disulfide cyclopeptide coding sequence. In the invention, it is found for the first time that a high glutathione (GSH) environment in a tumor environment can reduce and break a disulfide cyclopeptide of a linker polypeptide with a specific structure, so that the disulfide cyclopeptide is broken at a specific position, and the disulfide cyclopeptide is inserted into an N-terminal structural domain and a C-terminal structural domain of certain proteins, so that the disulfide cyclopeptide of the linker polypeptide with a specific structure is obtained. Specific response cutting can be carried out in a tumor environment, and protein functions are not affected, so that a unique anti-tumor effect is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to the application of disulfide cyclic peptides and biomaterials containing disulfide cyclic peptides in anti-tumor treatment. Background Art

[0002] Traditional cancer treatments, including chemotherapy and targeted therapies, have improved cancer patients' survival to a certain extent. However, these drugs still face significant toxic side effects, significantly reducing patients' quality of life. Furthermore, some patients develop drug resistance after long-term use, leading to a higher rate of tumor recurrence.

[0003] Since ipilimumab became the first immune checkpoint therapy drug approved by the US FDA, immune checkpoint therapy, a key branch of tumor immunotherapy, has entered a golden age of development. Immune checkpoint therapy has made significant progress in the treatment of melanoma, non-small cell lung cancer, and renal cancer, bringing new hope to cancer patients. Although this new anti-cancer strategy has made great progress in treating various solid tumors, in some tumors, such as pancreatic cancer, neither a single immune checkpoint inhibitor nor a combination of two immune checkpoint antibodies has achieved the desired therapeutic effect, with clinical trials even showing a 0% response rate.

[0004] Pyroptosis is a form of immune cell death characterized by cell membrane rupture. Pyroptosis can directly kill tumor cells or lead to an inflammatory response by releasing inflammatory factors and danger signaling molecules, thereby inducing the infiltration of cytotoxic T cells and activating T-cell-mediated anti-tumor immune responses. Studies have shown that inducing pyroptosis in tumor tissue can activate T cell immunity, and combined with immune checkpoint therapy, it can enhance anti-tumor function. Studies have also shown that inducing pyroptosis in 10-20% of tumor cells is sufficient to eliminate the entire tumor graft. Existing studies have also demonstrated that combining the activation of tumor cell pyroptosis with immune checkpoint therapy can significantly enhance the efficacy of immune checkpoint therapy, thereby promoting tumor regression and preventing tumor recurrence, providing another promising strategy for cancer treatment.

[0005] Gasdermin (GSDM) protein is the key executor of pyroptosis and the key factor that determines the occurrence of pyroptosis in cells. At present, the gasdermin proteins Gasdermin D and Gasdermin E and their role in pyroptosis have been studied relatively thoroughly. However, it should be noted that there are many limitations to initiating pyroptosis in tumor cells by cutting Gasdermin D and Gasdermin E: (1) Pyroptosis-related genes such as Gasdermin D and Gasdermin E are expressed in both normal tissues and tumor tissues. In some normal cells, their expression levels are even higher than those in some tumor cells. Therefore, inducing pyroptosis by general means may harm normal cells; (2) There are membrane repair molecular machines in cells that resist pyroptosis, namely, the ESCRT (essential endosomal sorting complex for transport) system shrinks the damaged plasma membrane at the membrane pores generated by GSDMD-N to complete membrane repair, thereby making pyroptosis ineffective; (3) Studies have shown that tumor cells can escape the occurrence of pyroptosis in different ways. Therefore, how to initiate pyroptosis in tumor cells without being restricted by the tumor itself has become the biggest challenge in realizing the use of pyroptosis for anti-tumor purposes.

[0006] Typically, a linker in a fusion protein maintains the conformation of the recombinant active protein or acts as a connecting peptide between proteins, enabling tandem expression of the component proteins without affecting their function. The choice of linker amino acid sequence and length is generally empirical, and currently no set rules exist.

[0007] Patent US20080299612 A1 discloses a linker sequence (LEAGCKNFFPRSFTSCGSLE (SEQ ID NO: 1)) containing a disulfide cyclic peptide and its preparation method. The patent primarily addresses the issue of improving the delivery efficiency and bioavailability of oral protein macromolecules, but does not explore the role of the linker in anti-tumor drugs. Furthermore, the patent describes the exogenous synthesis of a recombinant protein containing the linker and its use in the presence of exogenous thrombin, without discussing the specific effects of the linker inserted into the endogenous genome in the tumor microenvironment. Summary of the Invention

[0008] The present invention aims to address at least one of the aforementioned technical problems existing in the prior art. To this end, the present invention provides a disulfide cyclic peptide and a biomaterial containing the disulfide cyclic peptide for use in anti-tumor applications. The present invention, for the first time, discovered that the high glutathione (GSH) environment in the tumor environment can reduce and cleave the disulfide cyclic peptide of a specific linker polypeptide structure, thereby disconnecting it at a specific location. By inserting the disulfide cyclic peptide into the N-terminal and C-terminal domains of certain proteins, it can achieve specific responsive cleavage in the tumor environment without affecting protein function, thereby exerting a unique anti-tumor effect.

[0009] The first aspect of the present invention provides the use of a disulfide cyclic peptide in preparing a tumor microenvironment responsive element.

[0010] In some embodiments of the present invention, the disulfide cyclic peptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0011] In the present invention, "comprise", "contain", "have" or "include" means that at least the specified substance, component, element or method step is present in the product, article or method, but does not exclude the presence of other substances, components, elements, method steps, even if other such substances, components, elements, method steps have the same function as specified.

[0012] In some embodiments of the present invention, the concentration of glutathione (GSH) in the tumor microenvironment is 2-10 mM.

[0013] In some embodiments of the present invention, 10 mM GSH is used to simulate the high reducing properties in the tumor microenvironment.

[0014] In the present invention, the term "tumor microenvironment (TME)" refers to the dynamic and complex ecosystem in which tumor cells are located, which includes the local environment composed of tumor cells and non-tumor cells, interstitial components and biological molecules.

[0015] In some embodiments of the present invention, the tumor drug includes:

[0016] (1) biological material containing disulfide cyclic peptides or their coding sequences; and / or

[0017] (2) A reagent capable of inserting a disulfide cyclic peptide or its coding sequence into a specific position in a gene sequence.

[0018] In some embodiments of the present invention, the gene sequence includes any nucleic acid molecule that can trigger, promote or induce cell death or decreased vitality.

[0019] In some embodiments of the invention, the cells comprise tumor cells.

[0020] In some embodiments of the present invention, the gene sequence in (2) includes but is not limited to: the coding sequence of Gasdermin protein.

[0021] In some embodiments of the present invention, the gasdermin protein includes at least one of gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E.

[0022] In some embodiments of the present invention, the gasdermin protein includes at least one of gasdermin D and gasdermin E.

[0023] In some embodiments of the present invention, the gasdermin protein is gasdermin D or gasdermin E.

[0024] In some embodiments of the present invention, the position described in (2) includes at least one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein.

[0025] In some embodiments of the present invention, the one or more disulfide cyclic peptides or their coding sequences are inserted into at least one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein in a continuous or discontinuous manner.

[0026] In some embodiments of the present invention, the multiple disulfide cyclic peptides or their coding sequences are inserted into at least one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein in a continuous or discontinuous manner.

[0027] In some embodiments of the present invention, when the multiple disulfide cyclic peptides or their coding sequences are inserted into at least one side of the N-segment domain and / or the C-terminal domain of the Gasdermin protein in a non-contiguous manner, the multiple disulfide cyclic peptides or their coding sequences may be separated by: functional groups, including but not limited to biotin, digoxigenin, fluorescent groups, coupling sites (such as amino groups or sulfhydryl groups); chemical modifications, including but not limited to thiophosphates, photocleavable groups (PC linkers), cyclobutane groups, benzylamine groups, etc.; nucleotide sequences, including but not limited to hairpin structure sequences, double-stranded complementary regions, enzyme cleavage sites or functional aptamers; blocking groups, including but not limited to phosphorylation modifications and C3 Spacer.

[0028] In some embodiments of the present invention, the one or more disulfide cyclic peptides or their coding sequences are directly or indirectly connected to at least one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein.

[0029] In some embodiments of the present invention, the disulfide cyclic peptide or its coding sequence is directly or indirectly connected to one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein.

[0030] In some embodiments of the present invention, the disulfide cyclic peptide or its coding sequence is directly connected to one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein.

[0031] In some embodiments of the present invention, the disulfide cyclic peptide or its coding sequence is directly connected to the N-segment domain of the Gasdermin protein, or the disulfide cyclic peptide or its coding sequence is directly connected between the N-segment domain and the C-terminal domain of the Gasdermin protein.

[0032] In some embodiments of the present invention, the reagents described in (2) include reagents used in the following techniques:

[0033] CRISPR system, TALENs technology, megaTAL technology, DNA recombination technology ZFNs technology, Prime Editing technology, Cre-lox recombination system, homologous recombination and base precision insertion technology.

[0034] In some embodiments of the present invention, the precise base insertion technology includes single-base editors, PASTE and PGI.

[0035] In some embodiments of the present invention, the technology described in (2) is further used in combination with other technologies, including but not limited to homology repair (HDR), amplification (such as PCR, isothermal or constant temperature amplification, etc.), chain displacement, etc.

[0036] In some embodiments of the present invention, the biomaterial in (1) comprises:

[0037] (1) Disulfide cyclic peptides;

[0038] (2) a nucleic acid molecule encoding a disulfide cyclic peptide;

[0039] (3) an expression vector containing the nucleic acid molecule described in (2);

[0040] (4) A transformant containing the nucleic acid molecule described in (2);

[0041] (5) A transformant containing the expression vector described in (3).

[0042] In some embodiments of the invention, the nucleic acid molecules include DNA and RNA.

[0043] In some embodiments of the present invention, the DNA includes cDNA, ssDNA and dsDNA.

[0044] In some embodiments of the invention, the RNA comprises mRNA.

[0045] In some embodiments of the present invention, the transformants do not involve animal or plant reproductive materials.

[0046] In some embodiments of the present invention, the expression vector includes viruses, plasmids and mRNA.

[0047] In the present invention, the term "expressor" refers to a vector or expression system used to integrate or insert a foreign gene of interest.

[0048] In some embodiments of the present invention, the expression subunit further includes at least one of: a promoter, an enhancer sequence, a replication origin, a terminator, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, and a reporter gene.

[0049] In some embodiments of the present invention, the virus includes lentivirus, adenovirus, retrovirus and adeno-associated virus.

[0050] In some embodiments of the present invention, the transformants include bacteria (such as Escherichia coli or Bacillus subtilis, etc.), fungi (such as yeast or Aspergillus, etc.), and animal and plant cells.

[0051] In some embodiments of the present invention, the transformant further comprises insect cells, such as S2 Drosophila cells or Sf9 cells.

[0052] In the present invention, the term "transformant" refers to a recipient cell that has acquired a new genetic marker after incorporation or introduction of a foreign gene.

[0053] In some embodiments of the present invention, the construction of the expression vector and the transformant can be achieved based on any conventional technical means in the art.

[0054] In some embodiments of the present invention, the mRNA is as shown in SEQ ID NO:7.

[0055] In some embodiments of the present invention, the disulfide cyclic peptide has an amino acid sequence as shown in SEQ ID NO: 1.

[0056] In some embodiments of the present invention, the nucleic acid molecule encoding the disulfide cyclic peptide includes the nucleic acid molecule underlined in SEQ ID NO:6.

[0057] In some embodiments of the present invention, the disulfide cyclic peptide further comprises a functional group or a chemical modification.

[0058] In some embodiments of the present invention, the functional group includes at least one of a reporter group and a protein tag.

[0059] In the present invention, the position of the functional group is not limited. Based on actual use requirements and the specific selection of the functional group, it can be optionally inserted at any position on either end or in the middle of the fusion protein without significantly reducing at least one function of the fusion protein. In the present invention, the functions include anti-tumor activity, cleavage activity, and tumor microenvironment responsiveness.

[0060] In some embodiments of the present invention, the chemical modification comprises at least one of glycosylation, methylation, acetylation and phosphorylation.

[0061] In some embodiments of the present invention, the tumor comprises at least one of breast cancer, pancreatic cancer, melanoma, lung cancer, and glioma.

[0062] The beneficial effects of the present invention are:

[0063] (1) The disulfide cyclic peptide linker in the present invention can serve as a more specific response switch;

[0064] Through research, the present invention discovered the post-translational tertiary structure of the disulfide peptide linker, which is a disulfide peptide formed through an internal disulfide bond between cysteine ​​residues at positions 5 and 16. The high glutathione (GSH) environment in the tumor environment can reduce and cleave the disulfide peptide. As GSH continues to reduce the protein, it eventually breaks at arginine 11 and serine 12, thus independent of exogenous inducing agents. By inserting this linker into the N-terminal and C-terminal domains of a protein, specific cleavage can be achieved in the tumor environment without affecting protein function.

[0065] (2) The present invention provides a more precise transduction method, which can enable the linker to be precisely inserted into a specified genomic location;

[0066] The present invention utilizes gene editing and homologous recombination repair to accurately insert the linker into the specified position, and the linker does not rely on exogenous promoter expression, that is, the inserted gene can be regulated by the natural promoter of the inserted protein.

[0067] (3) The present invention provides a more stable transduction method;

[0068] Compared with the existing plasmid transient transfection technology, the gene knock-in technology used in the present invention does not have the defects of gradually losing the target characteristics or being unable to stably express during cell passage, and can achieve the effect of inserting the genome of tumor cell specific proteins and stably expressing them without being affected by cell passage.

[0069] (4) safe and versatile;

[0070] The technology in the present invention does not cause cell damage or pyroptosis to normal cells, and the precise knock-in technology can make its application scenarios more diverse.

[0071] (5) The present invention also provides a variety of proven effective biomaterials, such as a recombinant plasmid expressing a tumor microenvironment-responsive recombinant pyroptosis protein, which can release cytotoxic N-terminal pyroptosis protein with high specificity in the tumor microenvironment, effectively inducing tumor cell pyroptosis and improving the tumor immune microenvironment. Furthermore, it can be further based on a tumor-targeted fixed-point delivery tool to stably express the tumor microenvironment-responsive recombinant pyroptosis protein, reducing damage to normal cells, and is a safe and efficient protein drug delivery technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] Figure 1 Three disulfide cyclic peptide linkers are shown, wherein (a)-(c) correspond to SEQ ID NOs: 2, 3, and 1, respectively.

[0073] Figure 2 This is the map of plasmid VB240125-1643smg expressing sgRNA and Cas9.

[0074] Figure 3 This is the map of plasmid VB240125-1640khe that expresses the left and right homology arms of GSDME and the disulfide cyclic peptide linker.

[0075] Figure 4 Light microscopic images of B16F10 cells after transfection with different disulfide cyclic peptide linkers, where (a)-(c) correspond to SEQ ID NOs: 2, 3, and 1, respectively.

[0076] Figure 5 Light microscopic images of C2C12 cells after transfection with different disulfide cyclic peptide linkers, where (a)-(c) correspond to SEQ ID NOs: 2, 3, and 1, respectively.

[0077] Figure 6 Flow cytometry graphs of EMT-6 cells with and without the linker shown in SEQ ID NO: 1 inserted.

[0078] Figure 7Light microscopic images of EMT-6 cells with and without the linker shown in SEQ ID NO: 1 inserted.

[0079] Figure 8 The changes in tumor volume of mice in different experimental groups.

[0080] [[ID=⑧]]Figure 9 Actual photos of the changes in tumor volume of mice in different experimental groups.

[0081] [[ID=⑨]]Figure 10 Light microscopic images of 4T1 cells expressing and not expressing the linker shown in SEQ ID NO: 1.

[0082] [[ID=⑩]]Figure 11 The ribose gel electrophoresis results of recombinant engineered bacteria with different component compositions.

[0083] [[ID=⑪]]Figure 12 The changes in tumor volume of mice in different experimental groups.

[0084] [[ID=⑫]]Figure 13 Actual photos of the changes in tumor volume of mice in different experimental groups. DETAILED DESCRIPTION

[0085] The present invention is further described in detail below using specific examples. Unless otherwise specified, the raw materials, reagents, and apparatus used in the examples and comparative examples are available from conventional commercial sources or by conventional methods. Unless otherwise specified, all experimental or testing methods are conventional in the art.

[0086] Example 1

[0087] In this example, the effects of various disulfide cyclic peptides as linkers on the killing effect of tumor cells were tested. The specific experimental steps are as follows:

[0088] In this example, three disulfide cyclic peptide linkers were constructed, namely:

[0089] LEAGCKNFFPRSFTSCGSLE(SEQ ID NO: 1);

[0090] LEAGCKNGGGGSFTSCGSLE(SEQ ID NO: 2);

[0091] LEAGCKNFEAAAKTSCGSLE (SEQ ID NO: 3).

[0092] Among them, such as [[ID=⑬]]Figure 1 As shown in FIG, the above disulfide cyclic peptides can all be combined through the interaction of cysteines at positions 5 and 16 to form a disulfide bond, thereby forming a disulfide cyclic peptide.

[0093] Using CRISPR-Cas9 gene editing technology, two plasmids containing sgRNA and Cas9 protein (SaCas9), as well as left and right homology arms and the above-mentioned disulfide cyclic peptide linker were inserted: VB240125-1643smg and VB240125-1640khe (both plasmids were synthesized by Yunzhou Biotechnology Co., Ltd.).

[0094] The nucleotide sequence of VB240125-1643smg is:

[0095] 5'-CAACTTTGTATAGAAAAGTTGGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTG ACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCG [[ID=⑭]]GAGTTCGCCTTTCTGGACATGG [[ID=⑮]]TTTTAGTACTCTGGAAACAGAATCTACTAAAACAAGGCAAAATGCCGTGTTTATCTCGTCAACTTGTTGGCGAGATTTTTCAAGTTTGTACAAAAAAGCAGGCTGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCGCCACC [[ID=⑯]]ATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACG [[ID=⑰]]GAGTCCCAGCAGCCAAGCGGAACTACATCCTGGGCCTGGACATCGGCATCACCAGCGTGGGCTACGGCATCATCGA [[ID=⑱]]CTACGAGACACGGGACGTGATCGATGCCGGCGTGCGGCTGTTCAAAGAGGCCAACGTGGAAAACAACGAGGGCAGG [[ID=⑲]]CGGAGCAAGAGAGGCGCCAGAAGGCTGAAGCGGCGGAGGCGGCATAGAATCCAGAGAGTGAAGAAGCTGCTGTTCG [[ID=⑳]]ACTACAACCTGCTGACCGACCACAGCGAGCTGAGCGGCATCAACCCCTACGAGGCCAGAGTGAAGGGCCTGAGCCA [[ID=㉑]]GAAGCTGAGCGAGGAAGAGTTCTCTGCCGCCCTGCTGCACCTGGCCAAGAGAAGAGGCGTGCACAACGTGAACGAG [[ID=㉒]]GTGGAAGAGGACACCGGCAACGAGCTGTCCACCAAAGAGCAGATCAGCCGGAACAGCAAGGCCCTGGAAGAGAAAT [[ID=㉓]]ACGTGGCCGAACTGCAGCTGGAACGGCTGAAGAAAGACGGCGAAGTGCGGGGCAGCATCAACAGATTCAAGACCAG [[ID=㉔]]CGACTACGTGAAAGAAGCCAAACAGCTGCTGAAGGTGCAGAAGGCCTACCACCAGCTGGACCAGAGCTTCATCGAC [[ID=㉕]]ACCTACATCGACCTGCTGGAAACCCGGCGGACCTACTATGAGGGACCTGGCGAGGGCAGCCCCTTCGGCTGGAAGG [[ID=㉖]]ACATCAAAGAATGGTACGAGATGCTGATGGGCCACTGCACCTACTTCCCCGAGGAACTGCGGAGCGTGAAGTACGC CTACAACGCCGACCTGTACAACGCCCTGAACGACCTGAACAATCTCGTGATCACCAGGGACGAGAACGAGAAGCTG GAATATTACGAGAAGTTCCAGATCATCGAGAACGTGTTCAAGCAGAAGAAGAAGCCCACCCTGAAGCAGATCGCCA AAGAAATCCTCGTGAACGAAGAGGATATTAAGGGCTACAGAGTGACCAGCACCGGCAAGCCCGAGTTCACCAACCT GAAGGTGTACCACGACATCAAGGACATTACCGCCCGGAAAGAGATTATTGAGAACGCCGAGCTGCTGGATCAGATT GCCAAGATCCTGACCATCTACCAGAGCAGCGAGGACATCCAGGAAGAACTGACCAATCTGAACTCCGAGCTGACCC AGGAAGAGATCGAGCAGATCTCTAATCTGAAGGGCTATACCGGCACCCACAACCTGAGCCTGAAGGCCATCAACCT GATCCTGGACGAGCTGTGGCACACCAACGACAACCAGATCGCTATCTTCAACCGGCTGAAGCTGGTGCCCAAGAAG GTGGACCTGTCCCAGCAGAAAGAGATCCCCACCACCCTGGTGGACGACTTCATCCTGAGCCCCGTCGTGAAGAGAA GCTTCATCCAGAGCATCAAAGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAACGACATCATTATCGAGCT GGCCCGCGAGAAGAACTCCAAGGACGCCCAGAAAATGATCAACGAGATGCAGAAGCGGAACCGGCAGACCAACGAG CGGATCGAGGAAATCATCCGGACCACCGGCAAAGAGAACGCCAAGTACCTGATCGAGAAGATCAAGCTGCACGACA TGCAGGAAGGCAAGTGCCTGTACAGCCTGGAAGCCATCCCTCTGGAAGATCTGCTGAACAACCCCTTCAACTATGA GGTGGACCACATCATCCCCAGAAGCGTGTCCTTCGACAACAGCTTCAACAACAAGGTGCTCGTGAAGCAGGAAGAA AACAGCAAGAAGGGCAACCGGACCCCATTCCAGTACCTGAGCAGCAGCGACAGCAAGATCAGCTACGAAACCTTCA AGAAGCACATCCTGAATCTGGCCAAGGGCAAGGGCAGAATCAGCAAGACCAAGAAAGAGTATCTGCTGGAAGAACG GGACATCAACAGGTTCTCCGTGCAGAAAGACTTCATCAACCGGAACCTGGTGGATACCAGATACGCCACCAGAGGC CTGATGAACCTGCTGCGGAGCTACTTCAGAGTGAACAACCTGGACGTGAAAGTGAAGTCCATCAATGGCGGCTTCA CCAGCTTTCTGCGGCGGAAGTGGAAGTTTAAGAAAGAGCGGAACAAGGGGTACAAGCACCACGCCGAGGACGCCCT GATCATTGCCAACGCCGATTTCATCTTCAAAGAGTGGAAGAAACTGGACAAGGCCAAAAAAGTGATGGAAAACCAG ATGTTCGAGGAAAAGCAGGCCGAGAGCATGCCCGAGATCGAAACCGAGCAGGAGTACAAAGAGATCTTCATCACCC CCCACCAGATCAAGCACATTAAGGACTTCAAGGACTACAAGTACAGCCACCGGGTGGACAAGAAGCCTAATAGAGA GCTGATTAACGACACCCTGTACTCCACCCGGAAGGACGACAAGGGCAACACCCTGATCGTGAACAATCTGAACGGC CTGTACGACAAGGACAATGACAAGCTGAAAAAGCTGATCAACAAGAGCCCCGAAAAGCTGCTGATGTACCACCACG ACCCCCAGACCTACCAGAAACTGAAGCTGATTATGGAACAGTACGGCGACGAGAAGAATCCCCTGTACAAGTACTA CGAGGAAACCGGGAACTACCTGACCAAGTACTCCAAAAAGGACAACGGCCCCGTGATCAAGAAGATTAAGTATTAC GGCAACAAACTGAACGCCCATCTGGACATCACCGACGACTACCCCAACAGCAGAAACAAGGTCGTGAAGCTGTCCC TGAAGCCCTACAGATTCGACGTGTACCTGGACAATGGCGTGTACAAGTTCGTGACCGTGAAGAATCTGGATGTGAT CAAAAAAGAAAACTACTACGAAGTGAATAGCAAGTGCTATGAGGAAGCTAAGAAGCTGAAGAAGATCAGCAACCAG GCCGAGTTTATCGCCTCCTTCTACAACAACGATCTGATCAAGATCAACGGCGAGCTGTATAGAGTGATCGGCGTGA ACAACGACCTGCTGAACCGGATCGAAGTGAACATGATCGACATCACCTACCGCGAGTACCTGGAAAACATGAACGA CAAGAGGCCCCCCAGGATCATTAAGACAATCGCCTCCAAGACCCAGAGCATTAAGAAGTACAGCACAGACATTCTG GGCAACCTGTATGAAGTGAAATCTAAGAAGCACCCTCAGATCATCAAAAAGGGCAAAAGGCCGGCGGCCACGAAAA AGGCCGGCCAGGCAAAAAAGAAAAAGTACCCATACGATGTTCCAGATTACGCTTAAFigure 2 .

[0096] The nucleotide sequence of VB240125-1640khe is:

[0097] 5'- TCTTTCCTGTGCTCAACAAAAGGTGACTCCTCATCTCAGAAGACAAGGACACAGAGGGACCC CA AAGGCCCTCCTAAGCTTGCTGTAGTGCAGGACAAGTCTGTCAGCCACTCTATGCCTGATCAGACCTAGTGTAAGAG CCCTCAAGTGAGCAGACATGGTGGCATGTGTCTGTAATCCCAGCACGTGTGAGGCTGAGGCAGGAGGATCTGGGGT TAGAGATGAGCCTGGGCTACATAGTAAGACTCTATTAGTTATTTTTCTGTTGCTGTGACAAAGTACTGTGACCAAA GGCAACTCAAGGAAGAGTTCAATTTGGCTTATGGTTTCTGGGAGAGAAGAGTCGGCCATGGAGGGGCAGAGGCACG TCAGCAAGTGGCAGGCATGTGAGCGGGAGCAGGAAGCTGAGATCACACAGGGTGACAGTAGCTCAAAGGCTGTAAC TCAGAGCCCTCCCGCAGTGACTTACTTCCTCTAGCAGGACTGTACCTTCCAAAAAATGCCACTAAACTGGCGACTA AACACTCAAAAGCTGAGTCTATGGGAGATATTTCTCATTCAAACCACTAGACTCAGTCTCAAAAACTGATGCAAAG AAGCAAAAGCCCTTTTGTGTGTCTCCTGACACAGTGTGAGGTAGGTAACGATGAGTCTGAGGTGATCAGTGCCTGG CTTGGGCTTTTCTCTCCACTACAGAATTCTGCCTCCTCCAAGGGAAACATGGTGGCTTCGAGCATGAGAGGAAACT AGACTCTGTCTACTTGGACCCCCTGGCCTACAGAGAGTTCGCCTTTCTGGACATGCTTGACCTGGAGGCTGGGTGT AAAAACTTTTTTCCCCGATCTTTTACTTCGTGTGGGTCACTGGAGGGGGGTCAAGGGATCTCTTCGCAGGACGGGC CCCTACGAGTTGTAAAACAAGGTATTGCCTGTGGTTACGGCCAGATGGAGAAGTGGACTCTCTCCCAAGGGGTGAT CCCCTGGGGCCTTCAGGATCCCCTTGGATCTTTGGGTGGGCCCAGTCTTGGGTGTTGTGTTTTAGAGAATCGAGAT CCTAGGACTCAAGGTGGGAGGCCAAGGAGTGCAGTGGTCAGGGGCCTGACAGTTTTCTTTCCCCGTTTGGGACCTG TGGTCTGAGAATGGTCAGCAATGAGCAGAGCTAGAGCCCCCAGGATCTGTTATCAGCCTTGATACATTCCAGGCTT CACGGCATGGCAGGCTGCTGTTTGTAACAGCCATTGAGGCTTCCCACCCACATGGCTGGCGGAGGGCTGTAGCTCT GAACTATTGAGCAAACTCAATAGCAGTTTGCTTTGCTCCTCATAGCTTGTTACTTTAGGGTTTTTGTTTTGTTTTG TTTTGTTTTGTTTTGTTTTGCTGGGTTCAGGAGAGTGTGTGTGTGGGGGGAGCTGGGCTAGAAAAGGGGAGGAGCT TAGGAGAAACCAGGGCTGGCTCAAATTATGCTCTACCATGTAAAGCAATTCCTCCCTGGAATTCAGCAACTCTCTA ACCAGAGGATGCTGCTCCTGGCACAGTGTTAGAGGAAAGGGTGATGGAGACCCAGGGTTAGGGTAGCTTTTCCCTG GGATAAGAGTTAACTAAACTTGGGGCCTCTACTTGCAGTTGTGCTGCAGAAATGTGAGGCCACAGCTCCTTTTCTC ACTGCACCAFigure 3 .

[0098] The VB240125-1640khe plasmid (including the left homology arm-disulfide cyclic peptide linker coding sequence-right homology arm) was packaged and transfected into B16F10 cells (mouse skin melanoma cells) using lipo3000. The dosage of both plasmids was 2.5 μg / well, and the number of B16F10 cells was 2×10 6 Each well had a total volume of 2 mL / well. The culture medium used was RPMI-1640 medium supplemented with 10% fetal bovine serum. The cells were incubated at 37°C for 24 hours to complete the transfection. A nonsense plasmid control (PBS group) was also included.

[0099] The cells transfected with different disulfide peptide linkers were observed under an optical microscope. Figure 4 shown.

[0100] It can be found that the cells in the group in which only the disulfide cyclic peptide linker shown in SEQ ID NO: 1 was inserted showed obvious pyroptotic vesicles, while for the experimental groups in which the disulfide cyclic peptide linkers shown in SEQ ID NO: 2 and 3 were inserted, no pyroptotic vesicles were found at all, and no killing effect was observed.

[0101] Example 2

[0102] In this example, the cytotoxicity of the disulfide cyclic peptide linker was tested on normal cells using the following method:

[0103] According to the method in Example 1 above, the disulfide cyclic peptide linker was inserted into exon 5 of the GSDME genome of C2C12 cells (mouse myoblast cell line) using CRISPR-Cas9 gene editing technology. The dosage of both plasmids was 2.5 μg / well, and the number of C2C12 cells was 2×10 6 Each well was filled with 2 mL of DMEM supplemented with 5% fetal bovine serum. Transfection was completed by incubation at 37°C for 24 hours. A nonsense plasmid control (PBS group) was also included, and a positive control group (10 mM GSH) was added 6 hours after transfection to simulate the highly reducing microenvironment found in tumor tissue.

[0104] The results are as follows Figure 5 shown.

[0105] It can be found that when GSH is not added, no matter what kind of linker is inserted, normal cells will not produce pyroptosis. After the additional addition of GSH, only the group with the linker shown in SEQ ID NO: 1 inserted can cause pyroptosis of C2C12 cells, which once again verifies the results in Example 1 above, that is, it only responds to the tumor microenvironment.

[0106] Example 3

[0107] In this example, EMT-6 cells (mouse breast cancer cells) were used as the test object to test the effect of the above-mentioned disulfide cyclic peptide linker on the apoptosis of tumor cells. The test method is as follows:

[0108] According to the method in Example 1 above, two plasmids, SaCas9-sgRNA plasmid and donor plasmid (synthesized by Yunzhou Biotechnology Co., Ltd.), were packaged and transfected into EMT-6 cells using the Lipo 3000 kit (Thermo). The plasmids used, their dosages, and the total volume per well were the same as in Example 1. The number of EMT-6 cells was 2×10 5 The cells were transfected in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were incubated at 37°C for 12 hours to complete the transfection. A nonsense plasmid was also used as a control (PBS group).

[0109] After transfection, perform flow cytometry. Before performing flow cytometry, discard the liquid from the culture plate, wash the cells with PBS, and then digest them with trypsin. Resuspend the digested cells in PBS to obtain a cell suspension. Then, take 100 μL of the cell suspension and perform flow cytometric analysis using the Annexin V / PI method: add 5 μL of Annexin V / PI conjugate, incubate at room temperature for 15 minutes, then add 400 μL of 1× Annexin V binding solution, and analyze using a flow cytometer.

[0110] The results are as follows Figure 6 shown.

[0111] It can be found that after the linker shown in SEQ ID NO: 1 was knocked into the GSDME gene of tumor cells, the tumor cells showed obvious apoptosis.

[0112] Example 4

[0113] In this example, EMT-6 cells (mouse breast cancer cells) were used as the test subject to test the effect of the AAV vector based on the above-mentioned disulfide cyclic peptide linker on the pyroptosis of tumor cells. The test method is as follows:

[0114] According to conventional procedures in the art, two AAV vectors were constructed based on the above-mentioned SaCas9-sgRNA and donor plasmid frameworks for transfection (the inserted genes were the same as those in the above-mentioned VB240125-1643smg and VB240125-1640khe plasmids). The amount of the two AAV vectors used was 2×10 10 GC / mL, the number of EMT-6 cells was 2×10 6 Each well had a total volume of 2 mL / well. The culture medium used was RPMI-1640 medium supplemented with 10% fetal bovine serum. The cells were incubated at 37°C for 48 hours to complete the transfection. A nonsense plasmid control (PBS group) was also included.

[0115] The cells transfected with disulfide peptide linker were observed under an optical microscope. Figure 7 shown.

[0116] By knocking in the disulfide peptide linker shown in SEQ ID NO: 1 into aspartic acid at position 270 of the GSDME genome, the cells produced a large number of pyroptotic vesicles. Compared with the control group, it can be shown that knocking in the disulfide peptide linker shown in SEQ ID NO: 1 into GSDME can trigger pyroptosis of EMT-6 cells.

[0117] Example 5

[0118] In this example, tumor-bearing mice (EMT-6) were used as subjects to test the effect of the above-mentioned disulfide cyclic peptide linker on the pyroptosis of tumor cells. The testing method is as follows:

[0119] The two adeno-associated viruses constructed in Example 4 were mixed in equal proportions and co-administered as the GSDME-SS group, the empty adeno-associated virus was used as a negative control, and the PBS group was used as a blank control.

[0120] 5×10 5 EMT-6 cells were intratumorally administered on days 4, 7, and 10 after tumor inoculation. 8×10 10 Vg virus, tumor measurement was performed every two days to draw the tumor inhibition curve. The results are as follows Figure 8 The mice were euthanized at the end of treatment on day 23, and the tumors were removed and photographed. Figure 9 As shown in the figure, the tumor inhibition curve shows that the GSDME-SS group has a greater tumor inhibition effect, while there is no difference in tumor volume between the Ctrl and PBS groups. In vitro tumor images also demonstrate that only the group expressing the specific disulfide cyclic peptide has a significant tumor inhibition effect.

[0121] Example 6

[0122] Based on the anti-tumor effect of the disulfide cyclic peptide linker shown in SEQ ID NO: 1 and the GSDM protein found in the above examples, the inventors further designed a pyroptosis protein mRNA with tumor-specific killing effect, wherein the sequence of GSDME-SS mRNA is:

[0123] 5'-AUGUUUGCCAAAGCAACUCGAAAUUUUCUUAAAGAAGUUGAUGCUGGAGGAGACCUGAU UUCAGUCUCACACUUGAACGACUCUGACAAGCUGCAACUUCUAAGUCUGGGACCAAAAAGAAGAGAUACUGGUGCUGGCAGAGACCCAAGUACCAGAUUUUAUCUGCCACCCUGGAAGAUGUACUCACAGAAGGGCACUGUCUCAGUCCAGUGGUUGUGGAGUCAGACUUCGUGAAAUACGAGAGC AAGUGUGAGAACCAUAAGAGCGGGGCUAUUGGGACAGUCGUGGGGAAGGUCAAGCUGAACGUUGGUGGCAAAGGCGUGGUGGAGAGUCACUCUUCGUUUGGAACCCUGAGGAAGCAGGAGGUGGACGUGCAGCAGCUCAUCCAGGAUGCCGUCAAGAGAACAGUUAAUAUGGACAACCUGGUACUUCA GCAGGUGCUAGAGAGCAGGAACGAGGUCCUGUGUGUGCUGACGCAGAAGAUCAUGACCACGCAGAAGUGCGUGAUUUCUGAGCAUGUGCAGUCGGAGGACGUGUGGAGGCAUGGUGGGGAUCCAGACCAAGACUAUACAGGUGUCAGCAACGGAGGAUGGGACGGUCACCACGGACACCAAUGUAG UGCUGGAGAUCCCUGCUGCCACCACCAUUGCCUAUGGCAUCAUGGAGCUGUUUGUGAAACAAGAUGGCCAGUUUGAAUUCUGCCUCCCUCCAAGGGAAACAUGGUGGCUUCGAGCAUGAGAGGAAACUAGACUCUGUCUACUUGGACCCCCUGGCCUACAGAGAGUUCGCCUUUCUGGACAUGCUGGAU CUGGAGGCUGGGUG UAAAAACUUUUUUCCCCGAUCUUUUACUUCGUGUGGGUCACUGGAG-3' (SEQ ID NO: 6). The underlined portion is the coding sequence of the disulfide cyclic peptide linker shown in SEQ ID NO: 1, and the N-terminal domain and C-terminal domain of GSDME are before and after it.

[0124] The above mRNA sequence was delivered to a biological company for synthesis and construction of a recombinant plasmid containing the mRNA encoding GSDME-SS, using pUC-57 as the vector. The GSDME-SS sequence is:

[0125] 5’-ATGTTTGCCAAAGCAACTCGAAATTTTCTTAAAGAAGTTGATGCTGGAGGAGACCTGATTTCA GTCTCACACTTGAACGACTCTGACAAGCTGCAACTTCTAAGTCTGGTGACCAAAAAGAAGAGATACTGGTGCTGGCAGAGACCCAAGTACCAGATTTTATCTGCCACCCTGGAAGATGTACTCACAGAAGGGCACTGTCTCAGTCCAGTGGTTGTGGAGTCAGACTTCGTGAAATACGAGAGCAAGTGTGAGAACCATAAGAGCGGGGCTATTGGGACAGTCGTGGGGAAGGTCAAGCTGAACGTTGGTGGCAAAGGCGTGGTGGAGAGTCACTCTTCGTTTGGAACCCTGAGGAAGCAGGAGGTGGACGTGCAGCAGCTCATCCAGGATGCCGTCAAGAGAACAGTTAATATGGACAACCTGGTACTTCAGCAGGTGCTAGAGAGCAGGAACGAGGTCCTGTGTGTGCTGACGCAGAAGATCATGACCACGCAGAAGTGCGTGATTTCTGAGCATGTGCAGTCGGAGGAGACGTGTGGAGGCATGGTGGGGATCCAGACCAAGACTATACAGGTGTCAGCAACGGAGGATGGGACGGTCACCACGGACACCAATGTAGTGCTGGAGATCCCTGCTGCCACCACCATTGCCTATGGCATCATGGAGCTGTTTGTGAAACAAGATGGCCAGTTTGAATTCTGCCTCCTCCAAGGGAAACATGGTGGCTTCGAGCATGAGAGGAAACTAGACTCTGTCTACTTGGACCCCCTGGCCTACAGAGAGTTCGCCTTTCTGGACATGCTGGAT CTGGAGGCTGGGTG TAAAAACTTTTTTCCCCGATCTTTTACTTCGTGTGGGTCACTGGAG-3' (SEQ ID NO: 7). The underlined portion is the coding sequence of the disulfide cyclic peptide linker shown in SEQ ID NO: 1, and the N-terminal domain and C-terminal domain of GSDME are before and after it, respectively. The control group is wild-type GSDME mRNA that does not express SS.

[0126] The above mRNA sequence was delivered to a biosynthetic company to construct a recombinant plasmid containing the linker-pyroptosis protein N-terminal domain fusion protein sequence, using pUC-57 as a plasmid vector.

[0127] The mRNA extraction process is simplified as follows: BspQI enzyme (New England Biolabs) is used for enzymatic digestion in an ice-cooked reaction system. The reaction is incubated at 50°C for 5-15 minutes and inactivated at 80°C for 20 minutes to obtain circular DNA. DNA purification is performed using a kit (Monarch T1130S). Briefly, the sample is mixed with buffer BZ and centrifuged. DNA is adsorbed to the column and washed twice with buffer WZ. Elution is performed with buffer EY6 to obtain purified DNA. Using a kit (Monarch E2060S), the template mixture and kit components are mixed (incubated at 37°C for 30 minutes) and treated with DNase I for 15 minutes to remove the DNA template. Poly(A) tailing is then added (incubated at 37°C for 30 minutes, or up to 1 hour for short RNAs) to ensure 3'-end exposure, resulting in a crude RNA extract. Finally, buffer BX is added to bind the RNA with ethanol, followed by washing with buffer WX and elution with enzyme-free water to obtain purified mRNA.

[0128] GSDME mRNA and GSDME-SS mRNA were packaged and transfected into 4T1 cells (mouse breast cancer tumor cells) using lipo3000. The mRNA dosage was 5 μg / well and the number of 4T1 cells was 2×10 6 The total volume of each well was 2 mL / well. The culture medium used was RPMI-1640 medium containing 10% fetal bovine serum. Incubate at 37°C for 24 hours to complete the transfection. At the same time, a nonsense plasmid was set as a control (PBS group).

[0129] The cells after transfection with different mRNAs were observed using an optical microscope. Figure 10 shown.

[0130] It can be found that cells in the group (mRNA-GSDME-SS) in which only the disulfide cyclic peptide linker shown in SEQ ID NO: 1 was inserted showed obvious pyroptotic vesicles, while no pyroptotic vesicles were found in the group expressing wild-type GSDME, indicating that there was no killing effect on the cells.

[0131] Example 7

[0132] Based on the anti-tumor effect of the combination of the disulfide peptide linker shown in SEQ ID NO: 1 and the gasdermin protein found in the above examples, the inventors further designed a recombinant engineered bacterium for tumor treatment. The specific design steps are as follows:

[0133] In this example, Pseudomonas aeruginosa (PAO1) was used as the engineering bacterium, and the vector used was pS54-Cre, which was donated by Professor Bai Fang of Nankai University (reference for this vector is High-efficiency protein delivery into transfection-recalcitrant cell types. Biotechnology and bioengineering, 117(3), 816–831. https: / / doi.org / 10.1002 / bit.27245).

[0134]

[0135] To address the situation where the N-terminal domain may be buried when a protein tag (such as a flag, His tag, etc., see the sequence in SEQ ID NO: 8) is linked to the pyroptosis protein system, which may cause conformational changes and affect protein function, the disulfide cyclic peptide linker shown in SEQ ID NO: 1 can be inserted into the junction of the protein tag and the pyroptosis protein to obtain the linker-pyroptosis protein fusion protein sequence:

[0136] 5'- GACTACAAGGACGATGACGACAAG GAGCTCATG CTGGAGGCTGGGTGTAAAAACTTTTTTCCCCG ATCTTTTACTTCGTGTGGGTCACTGGAG

[0137] The recombinant plasmid containing the linker-pyroptosis protein N-terminal domain fusion protein sequence was constructed by Biosynthesis Company, using the pS54-Cre plasmid as the vector. The corresponding primers were designed:

[0138] Upstream primer F: 5′-AAAGGGGGATGTGCTGCAAGGCGATTAA-3′ (SEQ ID NO: 10);

[0139] Downstream primer R: 5′-GCTTCCGGCTCGTATGTTGTGTGGAATTG-3′ (SEQ ID NO: 11).

[0140] The results of ribose gel electrophoresis of successful polymerase chain reaction (PCR) construction are as follows Figure 11 shown.

[0141] The pS54-Cre plasmid, ligated with the target fragment, was transformed into DH5α competent cells, and positive clones were screened. Positive clones were verified using PCR and plasmid digestion, and amplified after confirmation. Plasmids were extracted from amplified positive clones using a commercially available kit and then electroporated into PAO1 for stable expression (alternatively, into E. coli Nissle 1917). Positive colonies were screened on plates containing the corresponding antibiotics to obtain an engineered bacterium expressing a tumor microenvironment-responsive recombinant pyroptosis protein with the tagged protein, named PAO1-SS-GSDMD.

[0142] Alternatively, without truncating the optimized sequence of the pyroptosis protein, a protein tag (such as a flag, His tag, etc., for its nucleotide sequence, see the corresponding portion of the sequence in SEQ IN NO: 9) is directly inserted before the N-domain of the pyroptosis protein to obtain a linker-pyroptosis protein fusion protein sequence. The above steps are then followed to amplify, recover the amplified fragment, perform enzyme digestion, ligate into a plasmid vector, transform cells, extract the plasmid, and then electroporate into PAO1 (or into EcN (E. coli Nissle 1917)) to obtain an engineered bacterium expressing a recombinant pyroptosis protein that does not respond to the tumor microenvironment and is expressed in a tagged protein, named PAO1-GSDMD.

[0143] After the above-mentioned engineered bacterial system is injected into tumor cells, it responds to the GSH reduction cleavage in the tumor environment to release cytotoxic pyroptosis protein. LPS on the surface of the bacterial carrier membrane activates caspase-1 to recognize the cleaved GSDMD sequence to obtain the cytotoxic N-terminus of the pyroptosis protein.

[0144] Therefore, the above schemes are not affected by the exogenous expression of various secretion systems. Various secretion systems can be freely assembled and expressed in series with pyroptosis proteins to initiate shearing in the tumor microenvironment.

[0145] In this example, tumor-bearing mice (4T1) were used as subjects to test the effect of the disulfide cyclic peptide linker on the pyroptosis of tumor cells. The testing method is as follows:

[0146] The two engineered bacteria PAO1-SS-GSDMD and PAO1-GSDMD constructed above were cultured in LB medium supplemented with D-glutamic acid until OD 600 When the value was 0.6, the culture was stopped, the mixture was centrifuged at 8000 rpm for 5 min, and the mixture was resuspended three times with PBS to obtain an engineered bacterial suspension for use.

[0147] 5×10 5 4T1 cells, when the tumor volume grows to 50-100mm 3 The tumor-bearing mice were injected intratumorally on days 0, 4, and 7, with 1×10 8 CFU engineered bacteria and tumors were measured every other day. The results were as follows Figure 12 shown.

[0148] At the end of treatment, mice were euthanized on day 14, and the tumors were removed and photographed. Figure 13 As shown in the figure, the tumor inhibition curve shows that the PAO1-SS-GSDMD group has a better tumor inhibition effect, and there is no difference in tumor volume between the PAO1-GSDMD group and the PBS group. In vitro tumor images also demonstrate that only the PAO1-S-GD engineered bacteria expressing the specific disulfide cyclic peptide have a significant tumor inhibition effect.

[0149] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of disulfide cyclic peptides in the preparation of tumor microenvironment-responsive elements; Preferably, the disulfide cyclic peptide has an amino acid sequence as shown in SEQ ID NO: 1; Preferably, the concentration of glutathione (GSH) in the tumor microenvironment is 2-10 mM.

2. Use of disulfide cyclic peptides in the preparation of anti-tumor drugs.

3. The use according to claim 2, characterized in that The tumor drugs include: (1) biological material containing disulfide cyclic peptides or their coding sequences; and / or (2) agents capable of inserting a disulfide cyclic peptide or its coding sequence into a specific position in a gene sequence; Preferably, the gene sequence includes any nucleic acid molecule that can trigger, promote or induce cell death or decreased vitality; Preferably, the cells comprise tumor cells.

4. The use according to claim 3, characterized in that The gene sequence includes the coding sequence of Gasdermin protein; Preferably, the gasdermin protein includes at least one of gasdermin A, gasdermin B, gasdermin C, gasdermin D and gasdermin E.

5. The use according to claim 3, characterized in that The locations mentioned in (2) include: At least one side of the N-terminal domain and / or the C-terminal domain of the Gasdermin protein and / or at least one gene site at the junction of the N-terminal domain and the C-terminal domain of the Gasdermin protein; Preferably, the one or more disulfide cyclic peptides or their coding sequences are inserted into at least one side of the N-terminal domain and / or C-terminal domain of the Gasdermin protein in a continuous or discontinuous manner; Preferably, the one or more disulfide cyclic peptides or their coding sequences are directly or indirectly connected to at least one side of the N-terminal domain and / or C-terminal domain of the Gasdermin protein.

6. The use according to claim 3, characterized in that The reagents described in (2) include reagents used in the following technologies: CRISPR system, TALENs technology, megaTAL technology, DNA recombination technology, ZFNs technology, PrimeEditing technology, Cre-lox recombination system, homologous recombination and base precision insertion technology; Preferably, the precise base insertion technology includes single-base editors, PASTE and PGI.

7. The use according to claim 3, characterized in that The biological materials mentioned in (1) include: (1) Disulfide cyclic peptides; (2) a nucleic acid molecule encoding a disulfide cyclic peptide; (3) an expression vector containing the nucleic acid molecule described in (2); (4) A transformant containing the nucleic acid molecule described in (2); (5) A transformant containing the expression vector described in (3); wherein the transformants do not involve animal or plant reproductive materials; Preferably, the nucleic acid molecules include DNA and RNA; Preferably, the DNA includes cDNA, ssDNA and dsDNA, and the RNA includes mRNA; Preferably, the expression vector includes viruses, plasmids and phages; Preferably, the transformants include bacteria, fungi, animals and plants.

8. The use according to claim 7, characterized in that The mRNA is shown in SEQ ID NO:

6.

9. The use according to claim 7, characterized in that The disulfide cyclic peptide has an amino acid sequence as shown in SEQ ID NO: 1; Preferably, the disulfide cyclic peptide further comprises a functional group or a chemical modification; Preferably, the functional group comprises: at least one of a reporter group and a protein tag; Preferably, the chemical modification comprises at least one of glycosylation, methylation, succinylation, palmitoylation, malonylation, acetylation and phosphorylation.

10. The use according to claim 1 or 2, characterized in that The tumor includes at least one of breast cancer, pancreatic cancer, melanoma, lung cancer and glioma.

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