Modular protein delivery system for targeted intracellular delivery of sustained-activation cGAS protein and its applications

Abstract

This invention belongs to the field of biopharmaceutical technology, specifically relating to a modular protein delivery system for targeted intracellular delivery of sustained-activation cGAS protein and its applications. The modular protein delivery system comprises a first functional module and a second functional module: the first and second functional modules are connected via a fragmentation-type intron or a Cage-Colockr system; the first functional module includes the cholera exotoxin B subunit or a functionally conserved variant thereof; the second functional module includes a sustained-activation cGAS mutant protein or a lysosomal degradation-resistant derivative thereof. The modular protein delivery system provided by this invention can be used to prepare immunotherapeutic drugs for the prevention or treatment of tumors, exhibiting advantages such as strong targeting and significant immune activation effects.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical technology, specifically relating to a modular protein delivery system for targeted intracellular delivery of sustained-activation cGAS protein and its application. Background Technology

[0002] The cGAS-STING signaling pathway is a crucial component of the innate immune system. Cytoplasmic DNA can be recognized by cGAS and catalyzed to generate the second messenger circular GMP-AMP (cGAMP), which in turn activates the STING signaling pathway, inducing the expression of type I interferon and various inflammatory factors, playing a key role in antiviral and antitumor immunity. Therefore, the regulation of the cGAS-STING pathway has become an important research direction in the field of tumor immunotherapy.

[0003] Current strategies for activating the cGAS-STING pathway mainly focus on exogenous cGAMP delivery or administration of small-molecule STING agonists. However, cGAMP and related small molecules typically have the following limitations: poor cell membrane permeability; limited in vivo stability, easily metabolized or cleared; and systemic administration may lead to abnormal activation of non-target tissue pathways, triggering inflammation-related adverse reactions. Furthermore, small-molecule agonists usually act on STING rather than cGAS, and cannot sustainably generate cGAMP within cells, thus limiting signaling persistence.

[0004] In contrast, direct delivery of cGAS protein with sustained catalytic activity to target cells can continuously generate cGAMP within the cell, activating the cGAS-STING pathway from its source, thereby achieving a more durable and controllable immune activation effect. However, this technical approach faces a dual challenge: on the one hand, cGAS is a large protein molecule, making it difficult to spontaneously cross the cell membrane into the cytoplasm; on the other hand, even if it enters the cell via endocytosis, it is easily degraded and inactivated by lysosomes, thus making it impossible to efficiently and controllably deliver continuously activated cGAS protein into target cells. Summary of the Invention

[0005] To achieve efficient and controllable delivery of continuously activated cGAS protein into target cells, this invention provides a modular fusion protein system for targeted delivery of continuously activated cGAS protein into cells and its application.

[0006] This invention provides a modular protein delivery system for targeted intracellular delivery of a sustained-activation cGAS protein, comprising a first functional module and a second functional module. The first and second functional modules are connected via a split intein system or a Cage-Colockr system. If the Cage-Colockr system is chosen, a third functional module, namely a tumor surface marker-specific binding domain, can be introduced to increase delivery selectivity. Furthermore, this invention uses a split intein system or a Cage-Colockr protein interaction system to connect different functional modules, rather than using direct gene fusion to construct the overall modular protein delivery system. The technical necessity lies in the fact that CtxB modules used to mediate endocytosis typically require inclusion body expression followed by denaturation-renaturation purification. However, this expression and purification method is not suitable for certain functional protein domains (e.g., some easily inactivated protein modules, those requiring native conformation, or those sensitive to folding environments). Direct fusion expression may lead to reduced expression efficiency, abnormal folding, or impaired function in the overall modular protein delivery system. By introducing a fragmented integrin system or a Cage-Colockr controlled assembly system, each functional module can be expressed and purified separately, allowing different modules to be obtained under their respective optimal expression and purification conditions, thus avoiding mutual interference. Subsequently, functional assembly between modules is achieved through specific protein recombination or protein-protein interactions.

[0007] The first functional module includes a cholera exotoxin B subunit (CtxB) or a functionally conserved variant thereof, wherein the cholera exotoxin B subunit can mediate protein endocytosis into the cell; wherein the amino acid sequence of the cholera exotoxin B subunit (CtxB) is shown in SEQ ID NO.3. The second functional module includes a persistently activating cGAS mutant protein (cGAS) or a lysosomal degradation-resistant derivative (cGAS LFmut), wherein the cGAS mutant protein can continuously catalyze the generation of cGAMP without DNA binding; wherein the amino acid sequence of the persistently activating cGAS mutant protein (referred to as cGAS mutant protein) is shown in SEQ ID NO.1.

[0008] The amino acid sequence of the lysosomal degradation-resistant derivative of the continuously activated cGAS mutant protein is shown in SEQ ID NO. 2.

[0009] The N-terminal fragment (CfaN) of the fracture-type intima-peptide is fused to the C-terminus of the first functional module, and the C-terminal fragment (CfaCC) of the fracture-type intima-peptide is fused to the N-terminus of the second functional module; wherein, the amino acid sequence of the fracture-type intima-peptide is as shown in SEQ ID NO.28; the N-terminal fragment of the fracture-type intima-peptide consists of amino acids 1 to 123, and its amino acid sequence is as shown in SEQ ID NO.4; the C-terminal fragment of the fracture-type intima-peptide consists of amino acids 124 to 158, and its amino acid sequence is as shown in SEQ ID NO.5.

[0010] The Cage-Colockr system is composed of the cage domain, Key domain and Bcl2 domain of amino acid sequences as shown in SEQ ID NO.6~SEQ ID NO.8, which are sequentially linked together. The cage domain is fused to the C-terminus of the first functional module and the Bcl2 domain is fused to the N-terminus of the second functional module.

[0011] The modular protein delivery system, after entering the cell via endocytosis, resides in the endoplasmic reticulum lumen through its KDEL sequence, continuously generating cGAMP, thereby activating the cGAS-STING signaling pathway.

[0012] This invention solves the technical problem of efficient and controllable delivery of continuously activated cGAS protein into target cells through modular design. Specifically, the first functional module uses the cholera exotoxin B subunit (CtxB) as a delivery carrier, utilizing its high affinity binding to GM1 gangliosides on the cell membrane to mediate the efficient entry of the protein complex into the cell via endocytosis, thereby overcoming the technical barrier of cGAS protein's inability to enter cells. The second functional module contains a continuously activated cGAS mutant protein or its lysosomal degradation-resistant derivative, ensuring that the delivered protein has stable catalytic activity in the cell and is not dependent on DNA-bound activation. The second functional module can also include endoplasmic reticulum resident sequences such as KDEL. To increase the probability of cGAS entering the endoplasmic reticulum lumen in the reverse transport pathway and enhance its stability, the two functional modules are connected via a fragmented integrin or a Cage-Colockr system. The former utilizes the intracellular self-splicing of fragmented integrins to form protein complexes, while the latter achieves spatiotemporally controllable protein complex formation through ligand-dependent assembly. Both mechanisms provide a modular paradigm for protein drug development. Furthermore, the Cage-Colockr system enables selective targeted delivery of cGAS protein, introducing a third functional module: the PD-L1 binding protein module. Thus, this modular delivery system simultaneously addresses three major technical bottlenecks: low cGAS protein entry efficiency, susceptibility to lysosomal degradation, and uncontrollable targeting, achieving efficient and controllable delivery of continuously activated cGAS protein into target cells.

[0013] Preferably, the second functional module further includes an endoplasmic reticulum resident signal peptide KDEL sequence, which is fused to the C-terminus of the continuously activated cGAS mutant protein or its lysosomal degradation-resistant derivative.

[0014] The amino acid sequence of the endoplasmic reticulum resident signal peptide KDEL is shown in SEQ ID NO.9.

[0015] Preferably, the first functional module further includes a targeting peptide fused to the N-terminus of the cholera exotoxin B subunit. The targeting peptide is an M2pep targeting peptide or other targeting peptides, and the amino acid sequence of the M2pep targeting peptide is shown in SEQ ID NO. 29: YEQDPWGVKWWY.

[0016] Preferably, in the Cage-Colockr system, the Key domain is used to fuse a targeting ligand, which is a single-chain variable fragment antibody (scFv) targeting PD-L1. The Key domain is fused with the single-chain variable fragment antibody targeting PD-L1 or other PD-L1 binding proteins (e.g., binding proteins obtained by generative AI) to form a Key-scFv modular protein delivery system.

[0017] The amino acid sequence of the single-chain variable fragment antibody targeting PD-L1 is shown in SEQ ID NO.10.

[0018] Preferably, the modular protein delivery system further includes an epitope tag, which is fused to the C-terminus of the second functional module.

[0019] The epitope tag is selected from HA tag, Flag tag, His6 tag, c-Myc tag or V5 tag, and the epitope tag can be integrated into the N-end, C-end or internal connection area of ​​each functional module.

[0020] Preferably, the lysosomal degradation-resistant derivative of the cGAS mutant protein is a mutant obtained by mutating phenylalanine at position 522 of the wild-type cGAS protein to alanine and leucine at position 523 to alanine. This mutant adds lysosomal resistance to the original characteristic of continuously catalyzing the generation of cGAMP without DNA binding.

[0021] The modular protein delivery systems for soluble expression include CfaCC-cGAS (as shown in SEQ ID NO.11), CfaCC-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.12), Bcl2-cGAS-Flag (as shown in SEQ ID NO.13), Bcl2-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.14), and Key-AtzscFv (as shown in SEQ ID NO.15).

[0022] Modular protein delivery systems expressed in the form of inclusion bodies include CtxB-HA-CfaN (as shown in SEQ ID NO.16), M2pep×2-CtxB-HA-CfaN (as shown in SEQ ID NO.17), and CtxB-HA-cage (as shown in SEQ ID NO.18).

[0023] The present invention also provides a protein pharmaceutical composition comprising the following components: Component A: The CtxB-HA-CfaN modular protein delivery system (as shown in SEQ ID NO.16) contains the cholera exotoxin B subunit (CtxB) and the fragmented inteptide N-terminal fragment (CfaN).

[0024] Component B: CfaCC-cGAS modular protein delivery system (as shown in SEQ ID NO.11), comprising a fractured inteptide C-terminal fragment (CfaCC) and a persistently activated cGAS mutant protein (cGAS).

[0025] When component A and component B are mixed, a complete CtxB-cGAS modular protein delivery system is formed through the trans-splicing activity of the fragmentation-type in-peptide.

[0026] Preferably, component B is a CfaCC-cGAS LFmut-Flag-KDEL modular protein delivery system (as shown in SEQ ID NO.12), wherein cGAS LFmut is a lysosomal degradation-resistant derivative with a Flag tag and KDEL sequence fused to its C-terminus.

[0027] The present invention also provides a protein drug composition targeting PD-L1 highly expressing cells, comprising the following components: The first component is the CtxB-cage modular protein delivery system, which contains the cholera exotoxin B subunit (CtxB) and the cage domain of the Cage-Colockr system.

[0028] The second component is the Bcl2-cGAS modular protein delivery system, which includes the Bcl2 domain of the Cage-Colockr system and the continuously activated cGAS mutant protein (cGAS) or its lysosomal degradation-resistant derivative (cGAS LFmut).

[0029] The third component is the Key-AtzscFv modular protein delivery system, which includes the Key domain of the Cage-Colockr system and a single-chain variable fragment antibody (AtzscFv) targeting PD-L1.

[0030] The first, second, and third components form a ternary complex on the surface of target cells through the specific interaction of the Cage-Colockr system, thereby achieving targeted delivery of cGAS protein to cells with high PD-L1 expression.

[0031] The present invention also provides a gene encoding the modular protein delivery system.

[0032] The present invention also provides an expression vector comprising the gene.

[0033] The present invention also provides a host cell containing the gene or the modular protein delivery system.

[0034] The present invention also provides the application of the modular protein delivery system, the gene, the expression vector, or the host cell in the preparation of cell biology tools for antitumor immune research.

[0035] The present invention also provides the use of the modular protein delivery system, the gene, the expression vector, or the host cell in the preparation of a drug for the prevention or treatment of tumors.

[0036] The cell biology tools described above for anti-tumor immunity research can be used to study the role and mechanism of the cGAS-STING signaling pathway in the regulation of the tumor immune microenvironment, including but not limited to: the effect of cGAS-STING pathway activation in tumor cells on tumor growth and metastasis; the regulatory role of cGAS-STING pathway activation in tumor-infiltrating immune cells (such as dendritic cells, macrophages, and T cells) on tumor immune response; and the interaction mechanism of the cGAS-STING pathway with other immune checkpoint molecules or signaling pathways.

[0037] The mechanism of action of the drug is as follows: through the modular protein delivery system, the continuously activated cGAS protein or its lysosomal degradation derivative is delivered to tumor cells or tumor-infiltrating immune cells, where it continuously catalyzes the generation of cGAMP intracellularly, thereby activating the cGAS-STING signaling pathway from the source, inducing the production of type I interferons (such as IFN-α, IFN-β) and pro-inflammatory cytokines (such as IL-6, TNF-α, CXCL10), thereby promoting dendritic cell maturation, enhancing antigen presentation, activating CD8⁺ T cell-mediated anti-tumor immune responses, and ultimately achieving the therapeutic effects of inhibiting tumor growth, metastasis, or eliminating tumors.

[0038] Preferably, if the drug is coupled to immune checkpoint-associated binding proteins via the Cage-Colockr system, it can also generate a synergistic anti-tumor effect. This not only prevents immune escape from tumor cells but also achieves targeted delivery to tumor cells.

[0039] Preferably, the tumor is selected from any one or more of melanoma, non-small cell lung cancer, breast cancer, colorectal cancer, liver cancer, and breast cancer. These tumor types all exhibit varying degrees of immune infiltration, and studies have shown that activation of the cGAS-STING pathway in their immune microenvironment is closely related to anti-tumor immune responses. Therefore, they are particularly suitable for targeted intervention using the modular protein delivery system of this invention.

[0040] Preferably, the drug can be administered via intravenous injection, subcutaneous injection, or intratumoral injection. Intratumoral injection can achieve local immune activation of the tumor and reduce systemic inflammatory adverse reactions; intravenous injection is suitable for systemic antitumor therapy; and subcutaneous injection is convenient for drug administration.

[0041] Preferably, the modular protein delivery system achieves the controlled release of continuously activated cGAS protein or its lysosomal degradation derivatives into target cells through the self-splicing of the fragmented intima-containing peptides or the ligand-dependent disassembly of the Cage-Colockr system.

[0042] Compared with the prior art, the present invention has the following beneficial effects: To achieve efficient and controllable delivery of continuously activated cGAS protein into target cells, this invention provides a modular protein delivery system for targeted intracellular delivery of continuously activated cGAS protein. The modular protein delivery system includes a first functional module and a second functional module: the first and second functional modules are connected via a fragmented integrin or a Cage-Colockr system; the first functional module includes a cholerae exotoxin B subunit or a functionally conserved variant thereof, which mediates protein endocytosis into the cell; the second functional module includes a continuously activated cGAS mutant protein or a lysosomal degradation-resistant derivative thereof, which continuously catalyzes the generation of cGAMP without DNA binding; the N-terminal fragment of the fragmented integrin is fused to the C-terminus of the first functional module, and the C-terminal fragment of the fragmented integrin is fused to the N-terminus of the second functional module; the Cage-Colockr system comprises a cage domain, a key domain, and a Bcl2 domain connected in sequence. This invention solves the technical problem of efficient and controllable delivery of continuously activated cGAS protein into target cells through modular design. Specifically, the first functional module uses the cholera exotoxin B subunit (CtxB) as a delivery carrier, leveraging its high affinity binding to GM1 gangliosides on the cell membrane to mediate the efficient entry of the protein complex into the cell via endocytosis, thus overcoming the technical barrier of cGAS protein's inability to enter cells. The second functional module contains a continuously activating cGAS mutant protein or its lysosomal degradation-resistant derivative, ensuring stable catalytic activity of the protein intracellularly after delivery, independent of DNA-bound activation. The second functional module can also include endoplasmic reticulum resident sequences such as KDEL. To increase the probability of cGAS entering the endoplasmic reticulum lumen in the reverse transport pathway and enhance its stability, the two functional modules are connected via a fragmented integrin or a Cage-Colockr system. The former utilizes the intracellular self-splicing of fragmented integrins to form protein complexes, while the latter achieves spatiotemporally controllable protein complex formation through ligand-dependent assembly. Both mechanisms provide a modular paradigm for protein drug development. Furthermore, the Cage-Colockr system enables selective targeted delivery of cGAS protein, introducing a third functional module: the PD-L1 binding protein module. Thus, this modular delivery system simultaneously addresses three major technical bottlenecks: low cGAS protein entry efficiency, susceptibility to lysosomal degradation, and uncontrollable targeting, achieving efficient and controllable delivery of continuously activated cGAS protein into target cells.

[0043] Compared with the prior art, the present invention has at least the following technical significance: (1) The first proposal and realization of delivering continuously activated cGAS protein as a functional molecule into tumor cells or immune-related cells to activate the cGAS-STING signaling pathway at its source.

[0044] (2) A modular assembly system suitable for intracellular delivery of macromolecular proteins was constructed, which solved the problem of incompatibility between expression and purification conditions of different protein domains.

[0045] (3) The targeted selection of delivery objects can be achieved through the interface of the replaceable target module.

[0046] (4) A scalable protein delivery platform has been formed, which can replace functional modules or targeting modules as needed for different treatment scenarios in order to achieve personalized cancer treatment.

[0047] Therefore, this invention not only provides a new tool for functional studies of the cGAS-STING pathway, but also offers a new approach to protein drug design for tumor immunotherapy. Attached Figure Description

[0048] Figure 1 This is a schematic diagram of the Western Blot detection results in this invention, used to verify that the CtxB-HA-CfaN+CfacC-cGAS modular protein delivery system can be effectively delivered into cultured cells.

[0049] Figure 2 This is a schematic diagram of the immunofluorescence staining results in this invention, used to verify that the CtxB-HA-CfaN+CfacC-cGAS-KDEL modular protein delivery system can successfully enter the cell.

[0050] Figure 3 This is a schematic diagram of the immunofluorescence staining results in this invention, used to verify that the Cage-Colockr-mediated cGAS protein delivery system can effectively deliver cGAS protein into the cell.

[0051] Figure 4 This is a schematic diagram of the functional verification results in this invention, used to demonstrate that the CtxB-HA-CfaN+CfacC-cGAS complex can activate related immune signaling pathways after being delivered into cells; wherein: A represents the detection of Tnf-α mRNA expression level after delivery; B represents the detection of the content of 2',3'-cGAMP small molecules in the cell culture medium after delivery; C represents the detection of Ifn-β1 mRNA expression level after delivery; Figure 5 This is a schematic diagram of the functional comparison results in this invention, used to verify that the CfacC-cGAS LFmut mutant can generate a more durable downstream immune signal response compared to CfacC-cGAS.

[0052] Figure 6This is a schematic diagram of animal experimental results in this invention, used to verify that the CtxB-cGAS delivery system has an inhibitory effect on tumor growth in animal models; wherein: A is a growth curve of tumor volume over time, showing the dynamic changes in tumor volume in different treatment groups during drug administration. Image B shows the image of the subcutaneous tumor removed at the experimental endpoint, corresponding to... Figure 6 A shows the two treatment conditions. Each tumor tissue sample in the figure represents an independent biological replicate.

[0053] Figure 7 This is a cartoon illustration of the delivery mode in this invention; wherein: A represents a protein assembly system mediated by the fragmentation of internal peptides; B represents a Cage-Colockr-mediated modular protein delivery system. Detailed Implementation

[0054] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0055] This invention provides a modular protein delivery system for targeted intracellular delivery of continuously activated cGAS protein, comprising a first functional module and a second functional module: the first functional module and the second functional module are connected by a fragmented integrin or a Cage-Colockr system.

[0056] The first functional module includes a cholera exotoxin B subunit or a functionally conserved variant thereof, wherein the cholera exotoxin B subunit is capable of mediating protein endocytosis into the cell; wherein the amino acid sequence of the cholera exotoxin B subunit (CtxB) is as shown in SEQ ID NO.3: TPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMAN.

[0057] The second functional module includes a continuously activating cGAS mutant protein or its lysosomal degradation-resistant derivative, wherein the cGAS mutant protein can continuously catalyze the generation of cGAMP without DNA binding; wherein the amino acid sequence of the cGAS mutant protein is as shown in SEQ ID. Shown in NO.1: SPDKLKKVLDKLRLDPKDISEAAAETVNKVVERLLRRMQKRESEFKGVEQLFVGSIYEKVKISAPNEFDVMFKLEVPRIELQEYYETGAFYLVKFKRIPRGNPLSHFLEGEVLSATKMLSKFRKIIKEEVKEIKDIDVSVEKEKPGSPAVTLLIRNPEEISVDIILALESKGSWPISTK EGLPIQGWLGTKVRTNLRREPFYLVPKNAKDGNSFQGETWRLSFSHTEKYILNNHGIEKTCCESSGAKCCRMECLMLMKYLLEQLKKEFQELDAFCSYHVKTAIFHMWTQDPQDSQWDPRNLSSCFDKLLAFFLECLRTEKLDHYFIPKFNLFSQELIDRKSKEFLSKKIEYERNNGFPIFDKL.

[0058] The amino acid sequence of the lysosomal degradation-resistant derivative of the cGAS mutant protein is shown in SEQ ID NO.2: SPDKLKKVLDKLRLDPKDISEAAAETVNKVVERLLRRMQKRESEFKGVEQLFVGSIYEKVKISAPNEFDVMFKLEVPRIELQEYYETGAFYLVKFKRIPRGNPLSHFLEGEVLSATKMLSKFRKIIKEEVKEIKDIDVSVEKEKPGSPAVTLLIRNPEEISVDIILALESKGSWPISTKEGL PIQGWLGTKVRTNLRREPFYLVPKNAKDGNSFQGETWRLSFSHTEKYILNNHGIEKTCCESSGAKCCRMECLMLMKYLLEQLKKEFQELDAFCSYHVKTAIFHMWTQDPQDSQWDPRNLSSCFDKALAAFLECLRTEKLDHYFIPKFNLFSQELIDRKSKEFLSKKIEYERNNGFPIFDKL.

[0059] The fragmented intima-intipeptide (amino acid sequence as shown in SEQ ID NO. 28) comprises an N-terminal fragment and a C-terminal fragment of the fragmented intima-intipeptide; the N-terminal fragment of the fragmented intima-intipeptide is fused to the C-terminus of the first functional module, and the C-terminal fragment of the fragmented intima-intipeptide is fused to the N-terminus of the second functional module; wherein, the amino acid sequence of the N-terminal fragment of the fragmented intima-intipeptide is shown in SEQ ID NO. 4: AEYCLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP.

[0060] The amino acid sequence of the C-terminal fragment of the fractured intima-intima peptide is shown in SEQ ID NO.5: VKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNC.

[0061] As shown in SEQ ID NO.28: AEYCLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLPVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNC.

[0062] The Cage-Colockr system comprises a cage domain, a key domain, and a Bcl2 domain connected in sequence; wherein the cage domain is fused to the C-terminus of the first functional module, the Bcl2 domain is fused to the N-terminus of the second functional module, and the key domain is used for fusion with a targeting ligand; the amino acid sequences of the cage domain, key domain, and Bcl2 domain are shown in SEQ ID NO. 6 to SEQ ID NO. 8, respectively. SEQ ID NO.6: LNSGSGSGKPGQASGSELARKLLEASTKLQRLNIRLAEALLEAIARLQELNLELVYLAVELTDPKRIRDEIKEVKDKSKEIIRRAEKEIDDAAKESEKILEEAREAISGSGSELAKLLLKAIAETQDLNLRAAKAFLEAAAKLQELNIRAVE LLVKLTDPATIREALEHAKRRSKEIIDEAERAIRAAKRESERIIEEARRLIEKGSGSGSELARELLRAHAQLQRLNLELLRELLRALAQLQELNLDLLRLASELTDEIWIAQELRRIGDEFNAYYADAERLSREAAAASEKISREAERLIR.

[0063] SEQ ID NO.7: GGSDEAIARVKRESKRIVEDAERLIREAAAASEKISREAERLIRGGGSGSGSGSGKPGQASGSDLGKKLLEAARAGQDDEVRIL.

[0064] SEQ ID NO.8: GSAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDDAEENRTEAPEGTESEVVHRALRDAGDDFERRYRRDFAEMSSQLHLTPDTARQRFETVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIAEWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRGEF.

[0065] Furthermore, the second functional module also includes an endoplasmic reticulum resident signal peptide KDEL sequence, the KDEL sequence being fused to the C-terminus of the continuously activated cGAS mutant protein or its lysosomal degradation-resistant derivative.

[0066] The amino acid sequence of the KDEL sequence is shown in SEQ ID NO.9: KDEL.

[0067] Furthermore, the first functional module may also include a targeting peptide fused to the N-terminus of the cholera exotoxin B subunit, wherein the targeting peptide is an M2pep targeting peptide or other targeting peptides.

[0068] The amino acid sequence of the M2pep targeting peptide is shown in SEQ ID NO. 29: YEQDPWGVKWWY.

[0069] Furthermore, in the Cage-Colockr system, the targeting ligand is a single-chain variable fragment antibody (scFv) targeting PD-L1, and the Key domain is fused with the single-chain variable fragment antibody targeting PD-L1 to form a Key-scFv modular protein delivery system.

[0070] The amino acid sequence of the single-chain variable fragment antibody targeting PD-L1 is shown in SEQ ID NO.10: MDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKREGKSSGSGSESK SEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS*.

[0071] Furthermore, the modular protein delivery system also includes an epitope tag, which is selected from HA tag, Flag tag, His6 tag, c-Myc tag or V5 tag.

[0072] Furthermore, the modular protein delivery system includes a modular protein delivery system for soluble expression and a modular protein delivery system for inclusion body expression.

[0073] The modular protein delivery systems for soluble expression include CfaCC-cGAS (as shown in SEQ ID NO.11), CfaCC-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.12), Bcl2-cGAS-Flag (as shown in SEQ ID NO.13), Bcl2-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.14), and Key-AtzscFv (as shown in SEQ ID NO.15).

[0074] Modular protein delivery systems expressed in the form of inclusion bodies include CtxB-HA-CfaN (as shown in SEQ ID NO.16), M2pep×2-CtxB-HA-CfaN (as shown in SEQ ID NO.17), and CtxB-HA-cage (as shown in SEQ ID NO.18).

[0075] The amino acid sequence of CfaCC-cGAS is shown in SEQ ID NO. 11: MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNCSPDKLKKVLDKLRLDPKDISEAAAETVNKVVERLLRRMQKRESEFKGVEQLFVGSIYEKVKISAP NEFDVMFKLEVPRIELQEYYETGAFYLVKFKRIPRGNPLSHFLEGEVLSATKMLSKFRKIIKEEVKEIKDIDVSVEKEKPGSPAVTLLIRNPEEISVDIILA LESKGSWPISTKEGLPIQGWLGTKVRTNLRREPFYLVPKNAKDGNSFQGETWRLSFSHTEKYILNNHGIEKTCCESSGAKCCRMECLMLMKYLLEQLKKEFQELDAFCSYHVKTAIFHMWTQDPQDSQWDPRNLSSCFDKLLAFFLECLRTEKLDHYFIPKFNLFSQELIDRKSKEFLSKKIEYERNNGFPIFDKLHHHHHH*.

[0076] CfacC is a component of the split intein system, used to undergo specific recombination reactions with the corresponding CfaN fragment, thereby enabling the connection and functional reconstruction of different protein fragments within the cell.

[0077] cGAS is an engineered cGAS protein that, after engineering modification, can continuously catalyze the production of cGAMP without DNA binding. The cGAS may be a variant containing specific functional mutation sites (e.g., enhanced stability or endoplasmic reticulum residency).

[0078] The modular protein delivery system can be used in combination with CtxB-HA-CfaN or M2pep×2-CtxB-HA-CfaN. Through a protein recombination reaction mediated by the fragmentation of integrins, a complete CtxB-fused cGAS protein is formed intracellularly, thereby achieving intracellular delivery of the cGAS protein or its selective delivery to M2 macrophages.

[0079] It should be understood that the above-described fusion construction method is not limited to the module connection method of the specific example. Equivalent adjustments made by those skilled in the art to the domain order, linker peptide length or connection method under the guidance of this invention should all fall within the protection scope of this invention.

[0080] The amino acid sequence of CfaCC-cGAS LFmut-Flag-KDEL is shown in SEQ ID NO.12: MVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASNCSPDKLKKVLDKLRLDPKDISEAAAETVNKVVERLLRRMQKRESEFKGVEQLFVGSIYEKVKISAPNEFD VMFKLEVPRIELQEYYETGAFYLVKFKRIPRGNPLSHFLEGEVLSATKMLSKFRKIIKEEVKEIKDIDVSVEKEKPGSPAVTLLIRNPEEISVDIILALESKGSWP ISTKEGLPIQGWLGTKVRTNLRREPFYLVPKNAKDGNSFQGETWRLSFSHTEKYILNNHGIEKTCCESSGAKCCRMECLMLMKYLLEQLKKEFQELDAFCSYHVKT AIFHMWTQDPQDSQWDPRNLSSCFDKALAAFLECLRTEKLDHYFIPKFNLFSQELIDRKSKEFLSKKIEYERNNGFPIFDKLHHHHHHHHYPYDVPDYAKDEL*.

[0081] CfacC is a component of the split intein system, used to undergo specific recombination reactions with the corresponding CfaN fragment, thereby enabling the connection and functional reconstruction of different protein fragments within the cell.

[0082] cGAS LFmut is an engineered cGAS mutant capable of continuously catalyzing cGAMP production without DNA binding. In this mutant, an amino acid substitution is made at one leucine (L) site and one phenylalanine (F) site, enhancing protein stability and sustained activity. It should be understood that the mutation sites are not limited to specific numbers; any equivalent mutant that achieves DNA-independent sustained catalytic activity enhancement or improved protein stability falls within the scope of this invention.

[0083] The flag is an epitope tag. In this modular protein delivery system, the flag tag is introduced solely for facilitating protein detection and tracing; it does not participate in functional performance. The epitope tag can be replaced with other commonly used epitope tags in the art, such as HA, V5, Myc, etc., or optionally, no epitope tag may be provided. Such substitutions or omissions do not affect the implementation of the technical solution or its functional effects. Therefore, the flag tag is not a necessary technical feature of this invention and is merely illustrative.

[0084] KDEL is a C-terminal endoplasmic reticulum (ER) retention signal sequence in mammalian cells, used to promote the retention of modular protein delivery systems in the ER region, thereby prolonging their functional duration within the cell. It should be understood that equivalent signal sequences capable of achieving similar ER retention functions can also replace the KDEL sequence (e.g., the HDEL sequence).

[0085] The modular protein delivery system can be used in combination with CtxB-HA-CfaN or M2pep*2-CtxB-HA-CfaN. Through a protein recombination reaction mediated by the fragmentation of integrins, a complete CtxB-fused cGAS protein is formed intracellularly, thereby achieving intracellular delivery of the cGAS protein or its selective delivery to M2 macrophages.

[0086] It should be understood that the above-described fusion construction method is not limited to the module connection method of the specific example. Equivalent adjustments made by those skilled in the art to the domain order, linker peptide length or connection method under the guidance of this invention should all fall within the protection scope of this invention.

[0087] The amino acid sequence of Bcl2-cGAS-Flag is shown in SEQ ID NO.13: *

[0088] Bcl2 is a component of the Cage-Colockr protein interaction system, specifically interacting with corresponding Cage modules to achieve controlled assembly between different modular protein delivery systems. Through this specific protein interaction, the directed binding and functional reconfiguration of multi-component modular protein delivery systems can be achieved on the cell surface or within the cell. Specifically, the Bcl2 domain can bind to modular protein delivery systems containing Cage modules, thereby enabling assembly with fusion systems containing targeting antibody fragments (e.g., the PD-L1 targeting antibody fragment AtzscFv) and CtxB protein.

[0089] cGAS is an engineered cGAS protein that, after engineering modification, can continuously catalyze the production of cGAMP without DNA binding. The cGAS may be a variant containing specific functional mutation sites (e.g., enhanced stability or endoplasmic reticulum residency).

[0090] The flag is an epitope tag. In this embodiment, the flag tag is introduced only to facilitate protein detection and tracing, and it does not participate in the functional process. The epitope tag can be replaced with other commonly used epitope tags in the art, such as HA, V5, Myc, etc., or the epitope tag can be omitted; the above substitution or omission does not affect the implementation of the technical solution of the present invention or its functional effect. Therefore, the flag tag is not a necessary technical feature of the present invention, but is only an illustrative example.

[0091] The modular protein delivery system can be used in combination with Key-AtzscFv and CtxB-HA-Cage. Assembly is completed on the target cell surface through specific protein interactions mediated by the Cage-Colockr system, thereby achieving selective delivery of cGAS protein to tumor cells expressing PD-L1 on their surface.

[0092] It should be understood that the arrangement order, connection method, and linker peptide length of the functional modules in the modular protein delivery system are not limited to the form shown in this embodiment. Equivalent structural adjustments or functional substitutions made by those skilled in the art under the guidance of this invention should all fall within the protection scope of this invention.

[0093] The amino acid sequence of Bcl2-cGAS LFmut-Flag-KDEL is shown in SEQ ID NO.14: *

[0094] Bcl2 is a component of the Cage-Colockr protein interaction system, specifically interacting with corresponding Cage modules to achieve controlled assembly between different modular protein delivery systems. Through this specific protein interaction, the directed binding and functional reconfiguration of multi-component modular protein delivery systems can be achieved on the cell surface or within the cell. Specifically, the Bcl2 domain can bind to modular protein delivery systems containing Cage modules, thereby enabling assembly with fusion systems containing targeting antibody fragments (e.g., the PD-L1 targeting antibody fragment AtzscFv) and CtxB protein.

[0095] cGAS LFmut is an engineered cGAS mutant capable of continuously catalyzing cGAMP production without DNA binding. In this mutant, an amino acid substitution is made at one leucine (L) site and one phenylalanine (F) site, enhancing protein stability and sustained activity. It should be understood that the mutation sites are not limited to specific numbers; any equivalent mutant that achieves DNA-independent sustained catalytic activity enhancement or improved protein stability falls within the scope of this invention.

[0096] The flag is an epitope tag. In this embodiment, the flag tag is introduced only to facilitate protein detection and tracing, and it does not participate in the functional process. The epitope tag can be replaced with other commonly used epitope tags in the art, such as HA, V5, Myc, etc., or the epitope tag can be omitted; the above substitution or omission does not affect the implementation of the technical solution of the present invention or its functional effect. Therefore, the flag tag is not a necessary technical feature of the present invention, but is only an illustrative example.

[0097] KDEL is a C-terminal endoplasmic reticulum (ER) retention signal sequence in mammalian cells, used to promote the retention of modular protein delivery systems in the ER region, thereby prolonging their functional duration within the cell. It should be understood that equivalent signal sequences capable of achieving similar ER retention functions can also replace the KDEL sequence (e.g., the HDEL sequence).

[0098] The modular protein delivery system can be used in combination with Key-AtzscFv and CtxB-HA-Cage. Through specific protein interactions mediated by the Cage-Colockr system, assembly is completed on the target cell surface, thereby achieving selective delivery of cGAS LFmut mutant protein to tumor cells expressing PD-L1 on the surface, and ensuring that the protein resides in the endoplasmic reticulum lumen after delivery.

[0099] It should be understood that the arrangement order, connection method, and linker peptide length of the functional modules in the modular protein delivery system are not limited to the form shown in this embodiment. Equivalent structural adjustments or functional substitutions made by those skilled in the art under the guidance of this invention should all fall within the protection scope of this invention.

[0100] The amino acid sequence of Key-AtzscFv is shown in SEQ ID NO.15: MGSSHHHHHHSSGGGSDEAIARVKRESKRIVEDAERLIREAAAASEKISREAERLIRGGGSGSGSGSGKPGQASGSDLGKKLLEAARAGQDDEVRILEFMDIQMTQSPSSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKREGKSSGSGSESKSEVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS*.

[0101] The Key domain is a component of the Cage-Colockr protein interaction system, specifically interacting with the corresponding Cage module to achieve controlled assembly between different modular protein delivery systems. Through this specific protein interaction, the directed binding and functional reconstruction of multi-component modular protein delivery systems can be achieved on the cell surface or within the cell. Specifically, the Key domain can bind to a modular protein delivery system containing a Cage module, thereby enabling the assembly of a fusion system containing a targeting antibody fragment and cGAS and CtxB proteins.

[0102] AtzscFv is a scFv antibody that targets PD-L1. It should be understood that any other antibody (such as a nanobody) or PD-L1 binding protein that can target PD-L1 can also be used as an alternative to AtzscFv.

[0103] The modular protein delivery system can be used in combination with CtxB-HA-Cage and Bcl2-cGAS LFmut-flag-KDEL or Bcl2-cGAS-flag. Through specific protein interactions mediated by the Cage-Colockr system, assembly is completed on the target cell surface, thereby achieving selective delivery of the cGAS LFmut mutant protein to tumor cells expressing PD-L1 on their surface, and ensuring that the delivered protein resides in the endoplasmic reticulum lumen.

[0104] The amino acid sequence of CtxB-HA-CfaN is shown in SEQ ID NO.16: MGSSHHHHHHTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANY PYDVPDYAAEYCLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP*.

[0105] CtxB is used to mediate the entry of modular protein delivery systems into cells via endocytosis.

[0106] CfaN is one of the two functional components of the split intein system, used to achieve the connection between the CtxB moiety and the cGAS protein moiety and subsequent recombination reaction, thereby forming a complete functional protein.

[0107] HA stands for epitope tag. The HA tag in this modular protein delivery system is only for facilitating protein detection and tracing; it does not participate in functional performance. The epitope tag can be replaced with other commonly used epitope tags in the art, such as Flag, V5, Myc, etc., or an epitope tag may be omitted; such substitution or omission does not affect the implementation of the technical solution or its functional effect. Therefore, the HA tag is not a necessary technical feature of this invention and is merely illustrative.

[0108] This protein can be used in combination with CfaC-cGAS or CfaC-cGAS LFmut-Flag-KDEL to form a complete CtxB fusion cGAS protein or cGAS LFmut mutant in the cell through a recombination reaction mediated by the breaking of the integrin, thereby achieving the delivery of the target protein into the cell.

[0109] The amino acid sequence of M2pep×2-CtxB-HA-CfaN is shown in SEQ ID NO.17: MYEQDPWGVKWWYGGGGSYEQDPWGVKWWYGGGGSGGGGSGGGGSGSSHHHHHHTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKV EKLCVWNNKTPHAIAAISMANYPYDVPDYAAEYCLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP*.

[0110] M2pep*2 is a tandem representation of the M2 macrophage-targeting peptide sequence, used to improve the selective recognition and binding affinity of the modular protein delivery system for M2 macrophages. It should be understood that the M2 macrophage-targeting peptide is not limited to the M2pep sequence; this invention also covers other functional targeting peptide sequences capable of achieving specific targeting of macrophage subsets. The above substitutions are equivalent transformations that can be achieved by those skilled in the art under the guidance of this invention, and all should fall within the protection scope of this invention.

[0111] CtxB is used to mediate modular protein delivery systems to enter cells via endocytosis.

[0112] CfaN is one of the two functional components of the split intein system, used to achieve the connection between the CtxB moiety and the cGAS protein moiety and subsequent recombination reaction, thereby forming a complete functional protein.

[0113] HA stands for epitope tag. The HA tag in this modular protein delivery system is only for facilitating protein detection and tracing; it does not participate in functional performance. The epitope tag can be replaced with other commonly used epitope tags in the art, such as Flag, V5, Myc, etc., or an epitope tag may be omitted; such substitution or omission does not affect the implementation of the technical solution or its functional effect. Therefore, the HA tag is not a necessary technical feature of this invention and is merely illustrative.

[0114] This protein can be used in combination with CfaC-cGAS or CfaC-cGAS LFmut-Flag-KDEL to form the complete cGAS protein or cGAS LFmut mutant in cells through a recombination reaction mediated by the breaking of the integrin, thereby achieving the delivery of the target protein into the interior of M2 macrophages.

[0115] The amino acid sequence of CtxB-HA-cage is shown in SEQ ID NO.18: MTPQNITDLCAEYHNTQIYTLNDKIFSYTESLAGKREMAIITFKNGAIFQVEVPGSQHIDSQKKAIERMKDTLRIAYLTEAKVEKLCVWNNKTPHAIAAISMANYP YDVPDYALNSGSGSGKPGQASGSELARKLLEASTKLQRLNIRLAEALLEAIARLQELNLELVYLAVELTDPKRIRDEIKEVKDKSKEIIRRAEKEIDDAAKESEKI LEAREAISGSGSELAKLLLKAIAETQDLNLRAAKAFLEAAAKLQELNIRAVELLVKLTDPATIREALEHAKRRSKEIIDEAERAIRAAKRESERIIEEARRLIEK GSGSGSELARELLRAHAQLQRLNLELLRELLRALAQLQELNLDLLRLASELTDEIWIAQELRRIGDEFNAYYADAERLSREAAAASEKISREAERLIRHHHHHH*.

[0116] CtxB is used to mediate the entry of modular protein delivery systems into cells via endocytosis.

[0117] HA stands for epitope tag. The HA tag in this modular protein delivery system is only for facilitating protein detection and tracing; it does not participate in functional performance. The epitope tag can be replaced with other commonly used epitope tags in the art, such as Flag, V5, Myc, etc., or an epitope tag may be omitted; such substitution or omission does not affect the implementation of the technical solution or its functional effect. Therefore, the HA tag is not a necessary technical feature of this invention and is merely illustrative.

[0118] Cage is a component of the Cage-Colockr protein interaction system, used to specifically interact with corresponding Key modules, thereby enabling controlled assembly between different modular protein delivery systems. Specifically, the Cage domain can mediate the binding between a modular protein delivery system containing CtxB and a modular protein delivery system containing a PD-L1 targeting antibody fragment (AtzscFv) and cGAS protein.

[0119] The modular protein delivery system can be used in combination with Key-AtzscFv and Bcl2-cGAS-Flag or Bcl2-cGASLFmut-Flag-KDEL. Assembly is completed on the target cell surface through specific protein interactions mediated by the Cage-Colockr system, thereby achieving selective delivery of cGAS protein or its LFmut mutant to tumor cells expressing PD-L1.

[0120] Example 1: Construction of expression plasmids for modular protein delivery system: 1. Gene synthesis and vector selection Based on the amino acid sequences shown in SEQ ID NO.11-SEQ ID NO.18, all modular protein delivery systems used prokaryotic expression vectors. The prokaryotic expression vector was the pET-28a(+) empty vector (pet28a vector).

[0121] CtxB-HA-CfaN (SEQ ID NO.16), M2pep*2-CtxB-HA-CfaN (SEQ ID NO.17), and CtxB-HA-cage (SEQ ID NO.18) were cloned into the prokaryotic pet28a vector, respectively, retaining the N-terminal signal peptide sequence to promote inclusion body formation, thus obtaining three different expression vectors.

[0122] The method for constructing the expression vector is as follows: 1.1 Design and synthesis of gene fragments: For the following three fusion proteins, the corresponding coding genes were designed and synthesized: CtxB-HA-CfaN, whose amino acid sequence is shown in SEQ ID NO.16.

[0123] M2pep*2-CtxB-HA-CfaN, whose amino acid sequence is shown in SEQ ID NO.17.

[0124] CtxB-HA-cage, whose amino acid sequence is shown in SEQ ID NO.18.

[0125] The gene fragments described above were provided by Azenta Life Science through artificial gene synthesis. The synthesized gene was cloned into a universal plasmid vector.

[0126] 1.2 Construction of the expression carrier: 1.2.1 Primer Design and PCR Amplification Using gene synthesis plasmids as templates, specific primers were designed to introduce homologous recombination arms at both ends of the gene fragment via PCR amplification. Forward homologous arm sequences (including) Nco The nucleotide sequence of the enzyme cleavage site and start codon is shown in SEQ ID NO.23: 5'-CCCCTCTAGAAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATG-3'.

[0127] The nucleotide sequence of the reverse homology arm (containing the His6 tag coding sequence and the stop codon) is shown in SEQ ID NO. 24: 5'-CTCAGCTTCCTTTCGGGCTTTGTTA GTGGTGGTGGTGGTGGTG-3'.

[0128] The reverse homology arm contains a His6 tag coding sequence of six consecutive histidine codons (CAT / CAC).

[0129] 1.2.2, Carrier linearization Using restriction endonucleases EcoR I (Beyotime, catalog number D6329) was used to perform single-enzyme digestion on the pET-28a(+) empty vector (Novagen, catalog number 69864-3) to obtain a linearized vector backbone. The digestion products did not require agarose gel electrophoresis separation and purification, and were directly added to the next reaction system for PCR ligation.

[0130] 1.2.3 PCR ligation cloning The insert fragment (containing the fusion protein coding sequence with homologous arms) obtained by PCR amplification was ligated in vitro with the EcoRI-linearized pET-28a vector backbone using PCR ligation to construct a recombinant expression vector. This design allows the C-terminus of the fusion protein, rather than the N-terminus, to carry a His6 tag for purification with TALON affinity resin (Takara Bio, catalog number 635502), thereby reducing the impact of the additional amino acid sequence on the function of M2pep and CtxB.

[0131] 1.2.4 Transformation and Screening The recombinant ligation product was transformed into DH5α competent Escherichia coli (Solarbio, catalog number C1100). The transformed bacterial culture was plated on LB solid medium containing kanamycin (50 μg / mL) and incubated at 37°C for 15 hours.

[0132] Single colonies were selected for colony PCR identification to screen for positive clones. Positive clones were further inoculated into LB liquid medium (containing 50 μg / mL kanamycin), cultured at 37°C with shaking, and then plasmids were extracted and verified by Sanger sequencing to ensure the correctness of the inserted sequence and the accuracy of the reading frame.

[0133] 1.2.5 Obtaining the Expression Vehicle The recombinant plasmids with verified correct sequences were extracted and purified using a plasmid miniprep kit to obtain the following expression vectors: pET-28a-CtxB-HA-CfaN expression vector.

[0134] pET-28a-M2pep*2-CtxB-HA-CfaN expression vector.

[0135] pET-28a-CtxB-HA-cage expression vector.

[0136] The expression vectors obtained above were stored at -20℃ for later use. These expression vectors are also referred to as prokaryotic expression plasmids encoding modular protein delivery systems.

[0137] CfaCC-cGAS (SEQ ID NO.11), CfaCC-cGAS LFmut-flag-KDEL (SEQ ID NO.12), Bcl2-cGAS-flag (SEQ ID NO.13), Bcl2-cGAS LFmut-flag-KDEL (SEQ ID NO.14), and Key-AtzscFv (SEQ ID NO.15) were cloned into the prokaryotic pet28a vector, and a His6 tag was introduced at the N-terminus to facilitate affinity purification, resulting in three different expression vectors.

[0138] The method for constructing the expression vector is as follows: 1.3 Design and synthesis of gene fragments: For each of the following five fusion proteins, the corresponding encoding genes were designed and synthesized: CfaCC-cGAS, whose amino acid sequence is shown in SEQ ID NO.11.

[0139] CfaCC-cGAS LFmut-flag-KDEL, whose amino acid sequence is shown in SEQ ID NO.12.

[0140] Bcl2-cGAS-flag, whose amino acid sequence is shown in SEQ ID NO.13.

[0141] Bcl2-cGAS LFmut-flag-KDEL, whose amino acid sequence is shown in SEQ ID NO.14.

[0142] Key-AtzscFv, whose amino acid sequence is shown in SEQ ID NO.15.

[0143] The gene fragments described above were provided by Azenta Life Science through artificial gene synthesis. The synthesized gene was cloned into a universal plasmid vector.

[0144] 1.4 Construction of the expression carrier: 1.4.1 Primer Design and PCR Amplification Using gene synthesis plasmids as templates, specific primers were designed to introduce homologous arms at both ends of gene fragments via PCR amplification. The nucleotide sequence of the forward homology arm (including the N-terminal His6 tag coding sequence and the start codon) is shown in SEQ ID NO. 25: 5'-GTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGC-3'.

[0145] The underlined portion represents the His6 tag coding sequence of six consecutive histidine codons (CAT / CAC).

[0146] The nucleotide sequence of the reverse homology arm (including the stop codon and the vector homology sequence) is shown in SEQ ID NO.26: 5'-TATGCTAGTTATTGCTCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTTGTTA-3'.

[0147] 1.4.2, Carrier Linearization Using restriction endonucleases EcoR I (Beyotime, catalog number D6329) was used to perform single-enzyme digestion on the pET-28a(+) empty vector (Novagen, catalog number 69864-3) to obtain a linearized vector backbone. The digestion product did not require agarose gel electrophoresis purification and was directly added to the next reaction system for PCR ligation.

[0148] 1.4.3 PCR ligation The insert obtained by PCR amplification (containing the fusion protein coding sequence of the N-terminal His6 tag and homologous arm) and... EcoR A linearized pET-28a vector backbone was used for in vitro ligation via PCR to construct a recombinant expression vector. This design enabled the fusion protein to carry a His6 tag at its N-terminus for protein purification using TALON affinity resin (Takara Bio, catalog number 635502).

[0149] 1.4.4 Transformation and Screening The recombinant ligation product was transformed into DH5α competent Escherichia coli (Solarbio, catalog number C1100). The transformed bacterial culture was plated on LB solid medium containing kanamycin (50 μg / mL) and incubated at 37°C for 14 hours.

[0150] Single colonies were selected for colony PCR identification to screen for positive clones. Positive clones were further inoculated into LB liquid medium (containing 50 μg / mL kanamycin), cultured at 37°C with shaking, and then plasmids were extracted and verified by Sanger sequencing to ensure the correctness of the inserted sequence and the accuracy of the reading frame.

[0151] 1.4.5 Obtaining the Expression Carrier The recombinant plasmids with verified correct sequences were extracted and purified using a plasmid miniprep kit to obtain the following expression vectors: pET-28a-His6-CfaCC-cGAS expression vector.

[0152] pET-28a-His6-CfaCC-cGAS LFmut-flag-KDEL expression vector.

[0153] pET-28a-His6-Bcl2-cGAS-flag expression vector.

[0154] pET-28a-His6-Bcl2-cGAS LFmut-flag-KDEL expression vector.

[0155] pET-28a-His6-Key-AtzscFv expression vector.

[0156] The expression vectors obtained above were stored at -20℃ for later use. These expression vectors are also referred to as prokaryotic expression plasmids encoding modular protein delivery systems.

[0157] 2. Design of key mutation sites The cGAS mutant protein was constructed based on engineered mouse cGAS, the amino acid sequence of which can be found in "Nature Structural & Molecular Biology 2023, 30, 72-80". This paper introduced the following key mutations: Continuous activation mutation: The E225A / D227A double mutation disrupts the DNA binding site, allowing cGAS to maintain its catalytic activity without DNA binding.

[0158] This invention further introduces a lysosomal degradation resistant mutation (LFmut): the L363A / F364A double mutation, which disrupts the lysosomal targeting sequence and prolongs the protein's half-life. This mutation was introduced via site-directed mutagenesis PCR and verified as correct by Sanger sequencing before being used in subsequent experiments.

[0159] The amino acid sequence of the cGAS mutant protein is shown in SEQ ID NO.1; the amino acid sequence of the lysosomal degradation-resistant derivative of the cGAS mutant protein is shown in SEQ ID NO.2.

[0160] Example 2: Induction and purification of modular protein delivery system 1. Protein purification methods The modular protein delivery system described in this embodiment was prepared using a prokaryotic expression system. The prokaryotic expression plasmid encoding the relevant modular protein delivery system (see Example 1) was transformed into Escherichia coli BL21(DE3) competent cells (Soleb, catalog number C1400) and amplified at 37°C. Subsequently, isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.5 mM was added at 16°C to induce expression. The bacterial cells were collected after induction.

[0161] The collected bacterial cells were resuspended in lysis buffer and sonicated on ice. They were then centrifuged at 13,000g for 10 minutes to obtain the supernatant and precipitate. Purification strategies were employed according to the expression state of different modular protein delivery systems. The lysis buffer consisted of 50mM NaH₂PO₄, 10mM imidazole, and 1% Triton X-100, pH 7.0.

[0162] 1) Purification methods for soluble proteins For modular protein delivery systems with soluble expression, including CfaCC-cGAS (as shown in SEQ ID NO.11), CfaCC-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.12), Bcl2-cGAS-Flag (as shown in SEQ ID NO.13), Bcl2-cGAS LFmut-Flag-KDEL (as shown in SEQ ID NO.14), and Key-AtzscFv (as shown in SEQ ID NO.15), purification was performed using a His6-tagged metal chelate affinity chromatography method. The specific steps are as follows:

[0163] Streptomycin sulfate to a final concentration of 2% (w / v) was added to the supernatant obtained after sonication lysis and centrifugation, and the mixture was stirred for 20 minutes to remove nucleic acid impurities. After centrifugation again, the supernatant was collected and incubated with TALON metal chelating resin (Takara Bio, catalog number 635502) at 4°C for 1 hour. The resin was then washed with washing buffer, and the target protein was eluted with elution buffer. The washing buffer consisted of 50 mM NaH₂PO₄, 30 mM imidazole, and 500 mM NaCl, pH 7.0. The elution buffer consisted of 50 mM aH₂PO₄, 300 mM imidazole, and 100 mM NaCl, pH 7.0.

[0164] The eluted protein was subjected to buffer replacement using Sephadex G-25 chromatography medium, transferred to storage buffer, and concentrated to obtain the protein stock solution, which was then stored at −80°C. The storage buffer consisted of 50 mM HEPES, 100 mM NaCl, and 10% glycerol.

[0165] 2) Purification and refolding methods for inclusion body proteins For modular protein delivery systems expressed in inclusion body form, including CtxB-HA-CfaN (as shown in SEQ ID NO. 16), M2pep×2-CtxB-HA-CfaN (as shown in SEQ ID NO. 17), and CtxB-HA-cage (as shown in SEQ ID NO. 18), affinity purification under denaturing conditions combined with gradient dialysis was used for refolding. The specific steps are as follows:

[0166] The precipitate obtained after sonication lysis and centrifugation was retained and resuspended in denaturing lysis buffer, with thorough stirring to dissolve the inclusion bodies. After another centrifugation, the supernatant was collected and incubated with TALON resin (Takara Bio, catalog number 635502) for 1 hour. The resin was then washed with the denaturing lysis buffer, and the target protein was eluted with denaturing elution buffer. The denaturing lysis buffer consisted of 100 mM NaH₂PO₄, 10 mM Tris, 20 mM imidazole, and 8 M urea, pH 8.0. The denaturing elution buffer consisted of 100 mM NaH₂PO₄, 300 mM Mimidazole, 8 M urea, and 10 mM Tris-HCl, pH 4.5.

[0167] The eluted protein was refolded by dialysis. A multi-step gradient dialysis process was used to gradually replace the protein from the elution buffer to the storage buffer. The dialysis was performed a total of 5 times, and the buffers used in each step were as follows: Buffer I: 50 mM Tris, 50 mM NaCl, 4 M urea, pH 7.5; Buffer II: 50 mM Tris, 50 mM NaCl, 2 M urea, pH 7.5; Buffer III: 50 mM Tris, 50 mM NaCl, 1 M urea, pH 7.5; Buffer IV: 50 mM Tris, 50 mM NaCl, 0.5 M urea, pH 7.5.

[0168] The refolded protein was further concentrated to obtain a protein stock solution, which was then stored at −80°C.

[0169] Example 3: Verification of intracellular delivery efficiency of CtxB-cGAS protein 1. Experimental grouping and treatment Human glioblastoma cell line U87 (U87 cells) was used as the model cell, and the experimental groups were as follows: Experimental group: CtxB-HA-CfaN and CfaCC-cGAS mixed (final concentration of each 0.2 μM).

[0170] Positive control group: cGAS-flag plasmid transfected with Lipofectamine 2000.

[0171] Negative control group: treated with PBS.

[0172] The modular protein delivery system was added to complete culture medium (containing 10% (w / v) FBS, 10% (w / v) NEAA and penicillin and streptomycin in DMEM) and incubated with cells at 37°C and 5% CO2 for 3 hours.

[0173] Among them, the human glioblastoma cell line U87 came from the National Experimental Cell Resource Sharing Platform.

[0174] The cGAS eukaryotic expression plasmid with the Flag tag (cGAS-flag plasmid) was obtained through homologous recombination cloning.

[0175] The cGAS-Flag plasmid cloning method is as follows: Using the above-mentioned CfaCC-cGAS pet28a expression plasmid as a template, fragments with homologous arms were obtained by extending the DNA fragment of the continuously activated cGAS using PCR. The N-terminal sequence of the cGAS fragment after homologous arm extension is ACCTCGAGCTCAAGCTTCGAATTCGCCACC (as shown in SEQ ID NO. 30), and the C-terminal sequence is GGATCCGACTACAAGGATGACGATGACA (as shown in SEQ ID NO. 31). The plenti-GFP-Flag vector (Heyuan Biotechnology Co., Ltd., catalog number H128) was obtained by double digestion with BamHI (Beyotime, catalog number D6053) and EcoRI (Beyotime, catalog number D6329) restriction endonucleases. The linearized backbone vector was then ligated to the cGAS insert fragment using a recombinase (TransGen, catalog number CU201-02). The obtained plasmid was verified to be correct by Sanger sequencing and used for subsequent experiments.

[0176] The sequence of the DNA fragment encoding the cGAS-flag plasmid is shown in SEQ ID NO.27:

[0177] The chemical composition of PBS is: NaCl 136.9 mM, KCl 2.68 mM, NaH2PO4 10.14 mM. With K2HPO4 1.8 mM, pH=7.4.

[0178] 2. Western blot detection After incubation, wash cells three times with pre-cooled PBS, add RIPA lysis buffer containing protease inhibitors, and lyse on ice for 30 minutes. Centrifuge at 10000g for 10 minutes, collect the supernatant for SDS-PAGE, transfer to a membrane, and incubate sequentially with anti-Flag antibody (1:1000, Invitrogen, PA-1-984B) or His6 antibody (1:1000, Proteintech, 66005-1-Ig) and HRP-labeled secondary antibody. Develop with ECL chemiluminescence.

[0179] Western blot results are as follows Figure 1 As shown, the experimental group exhibited a distinct cGAS band at approximately 58 kDa, corresponding to the molecular weight of CtxB+cGAS, while the negative control group showed no signal. This result confirms that the CtxB-cGAS modular protein delivery system, formed through fragmented inteptide transsplicing, can effectively enter U87 cells, with delivery efficiency comparable to liposome transfection.

[0180] As can be seen from the above, breaking the peptide-mediated protein linkage system can generate a modular protein delivery system CtxB-cGAS and mediate the entry of cGAS into the cell.

[0181] Example 4: Verification of endoplasmic reticulum colocalization of CtxB-cGAS-KDEL 1. Immunofluorescence staining HEK293A cells were seeded in confocal culture dishes and incubated for 3 hours with CtxB-cGAS-KDEL modular protein delivery system (final concentration 0.2 μM). After washing with PBS, the cells were fixed with 4% (v / v) paraformaldehyde for 15 minutes, permeabilized with 0.1% (w / v) Triton X-100 for 10 minutes, and blocked with 5% (w / v) BSA for 30 minutes.

[0182] Then add the following substances in sequence: Primary antibodies: anti-Flag antibody (Proteintech, catalog number 66008-4-Ig, 1:200, labeled cGAS) and anti-PDI antibody (Beyotime, catalog number AF0264, 1:200, labeled endoplasmic reticulum), incubated overnight at 4°C.

[0183] Secondary antibodies: Alexa Fluor 488-labeled anti-mouse IgG (green, cGAS) and Alexa Fluor 594-labeled anti-rabbit IgG (red, endoplasmic reticulum), 1 hour at room temperature.

[0184] After DAPI staining, the slides were mounted with anti-fluorescence quenching mounting medium and observed using a laser confocal microscope (Zeiss LSM 800).

[0185] HEK293A cells were purchased from Thermo Fisher Scientific. Overnight refers to a time of ≥12 hours.

[0186] The results are as follows Figure 2 As shown, the red fluorescence and the green part are co-located.

[0187] As can be seen from the above, the protein linkage system mediated by the breakage of intima-containing peptides can generate a modular protein delivery system CtxB-cGAS and mediate the entry of cGAS into the cell interior, where some reside in the endoplasmic reticulum.

[0188] Example 5: Validation of the Cage-Colockr-cGAS system targeting PD-L1 highly expressed cells 1. Obtain a cell population containing a portion of transiently overexpressing PD-L1. The lentiviral expression plasmid (plenti-PD-L1-Flag) expressing PD-L1 protein driven by the EF1α promoter was transfected into HEK293A cells using liposomes.

[0189] In this embodiment, the plenti-PD-L1-Flag can be found in "Nature Communications 2021, 12, 1940."

[0190] HEK293A cells were purchased from Thermo Fisher Scientific.

[0191] 2. Ternary system assembly and delivery The following components were added to the culture medium of HEK293A cells overexpressing PD-L1: CtxB-HA-cage (0.1μM); Bcl2-cGAS-flag (0.1μM); Key-AtzscFv (0.1μM).

[0192] Incubate at 37°C for 3 hours, and set up a control group lacking Key-AtzscFv.

[0193] 3. Immunofluorescence detection After fixation and permeabilization, the cells were incubated sequentially with anti-Flag antibody (1:200, Proteintech, catalog number 66008-4-Ig, for detecting cGAS) and anti-PD-L1 antibody (1:200, Abcam, catalog number Ab205921), followed by confocal imaging after fluorescent secondary antibody labeling.

[0194] See results Figure 3 .

[0195] In the Cage-Colockr-cGAS group, cells exhibiting green fluorescence (PD-L1 positive) always also showed red fluorescence. In the control group without Key-AtzscFv, cells exhibiting green fluorescence did not show red fluorescence.

[0196] As can be seen from the above, the cGAS-CtxB modular protein delivery system mediated by the Cage-Colockr system can selectively enter the interior of cells expressing PD-L1.

[0197] Example 6: Verification of cGAMP generation and downstream pathway activation after cGAS entry into cells 1. Cell processing and RNA extraction U87 cells were treated according to the method in Example 3, and cells were collected after 6 hours of incubation. Total RNA was extracted using TRIzol reagent and reverse transcribed into cDNA.

[0198] 2. qPCR detection of inflammatory factor expression The mRNA levels of the following genes were detected using the SYBR Green qPCR method: Ifn-β1 (Interferon-β1); Tnf-α (Tumor necrosis factor-α); GAPDH (Internal reference).

[0199] Primer sequences: Ifn-β1 The nucleotide sequence of -F is shown in SEQ ID NO.19: gcttggattcctacaaagaagca.

[0200] Ifn-β1 The nucleotide sequence of -R is shown in SEQ ID NO.20: atagatggtcaatgcggcgtc.

[0201] Tnf-α The nucleotide sequence of -F is shown in SEQ ID NO.21: gagcactgaaagcatgatcc.

[0202] Tnf-αThe nucleotide sequence of -R is shown in SEQ ID NO.22: cgagaagatgatctgactgcc.

[0203] 3. Quantitative detection of cGAMP Collect cell culture supernatant and use the 2'3'-cGAMP ELISA kit (Cayman Chem, catalog number 501700) to detect cGAMP concentration according to the manufacturer's instructions.

[0204] The results are as follows Figure 4 As shown: Compared with the control group (the NC group in the left and middle images or the control group CfaCC-cGAS without CtxB-CfaN in the right image), the CtxB-cGAS group showed higher expression levels of Tnf-α and Ifn-β1 and a higher concentration of 2',3'-cGAMP released into the culture medium.

[0205] The above results indicate that the cGAS protein delivered into the cell has catalytic activity, can continuously generate cGAMP and activate the downstream STING signaling pathway.

[0206] Example 7: Verification of cGAS-LFmut's resistance to lysosomal degradation and its sustained effect 1. Time gradient processing experiment U87 cells were divided into two groups, and CtxB-cGAS or CtxB-cGAS LFmut (final concentration 0.2 μM) were added to one group, and the other group was incubated at 37°C. Cells were collected at 0, 3, 6, 12, and 24 hours, and RNA was extracted for detection. Ifn-β1 mRNA levels were assessed to evaluate the activation level of the cGAS-STING signaling pathway.

[0207] The results are as follows Figure 5 As shown: Compared with the CtxB-cGAS treatment group, the CtxB-cGAS LFmut treatment group showed higher levels of Ifn-β1 mRNA expression at all time points. Even 24 hours after treatment, the Ifn-β1 expression level in the CtxB-cGAS LFmut group remained high, with no significant decreasing trend observed.

[0208] The results indicate that cGAS-LFmut has higher stability than the original cGAS, thereby inducing a more persistent downstream effect.

[0209] As shown above, both the CtxB-cGAS and CtxB-cGAS LFmut delivery systems mediated by the fragmentation inteptide system can achieve effective intracellular delivery and functional activation of cGAS protein; among them, cGAS LFmut shows better performance in terms of signal persistence.

[0210] Example 8: Animal experiments to verify the efficacy of tumor treatment 1. Establishment of a subcutaneous xenograft model Eight-week-old C57BL / 6 mice, half male and half female, were selected and subcutaneously inoculated with 5 × 10⁸ mice in the groin. 6 One Pan02 pancreatic cancer cell was inoculated. Ten days after inoculation, the cells were randomly divided into two groups (n=8 / group).

[0211] Control group: Intratumoral injection of PBS.

[0212] CtxB-cGAS group: Intratumoral injection of the CtxB-cGAS modular protein delivery system mediated by fragmented intima-containing peptides, with 25 μg of total protein per tumor.

[0213] The 8-week-old C57BL / 6 mice were obtained from Beijing Vital River Laboratory Technology Co., Ltd.

[0214] The CtxB-cGAS modular protein delivery system mediated by the fragmentation of the intrinating peptide is the modular protein delivery system prepared in Example 2, also known as the CtxB-cGAS modular protein delivery system.

[0215] 2. Dosing regimen The drug was administered once every 3 days, for a total of 3 administrations. Three days after the last administration, the mice were sacrificed, the tumor tissue was dissected, and the major axis (a) and minor axis (b) were measured. The tumor volume was calculated using the formula V = 0.5 × a × b², where V represents the tumor volume.

[0216] 3. Results are as follows Figure 6 As shown, the tumor volume in the CtxB-cGAS group was smaller than that in the control group.

[0217] As shown above, the CtxB-cGAS modular protein delivery system can inhibit tumor growth in subcutaneous xenograft models, suggesting its potential application value in tumor treatment.

[0218] And by Figure 7As can be seen, this invention employs a split intein system or a Cage-Colockr protein interaction system to connect different functional modules, rather than using direct gene fusion to construct a monolithic modular protein delivery system. The technical necessity lies in the fact that CtxB modules used to mediate endocytosis typically require inclusion body expression followed by denaturation-renaturation purification. However, this expression and purification method is not suitable for some functional protein domains (e.g., certain protein modules that are easily inactivated, require maintaining their native conformation, or are sensitive to their folding environment). Direct fusion expression may lead to reduced expression efficiency, abnormal folding, or functional impairment in the monolithic modular protein delivery system. By introducing a split intein system or a Cage-Colockr controllable assembly system, each functional module can be expressed and purified separately, allowing different modules to be obtained under their optimal expression and purification conditions, thus avoiding mutual interference. Subsequently, functional assembly between modules is achieved through specific protein recombination or protein interactions.

[0219] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A modular protein delivery system for targeted intracellular delivery of continuously activated cGAS protein, characterized in that, It includes a first functional module and a second functional module: the first functional module and the second functional module are connected by a fragmented inteptide or a Cage-Colockr system; The first functional module includes the cholera exotoxin B subunit or a functionally conserved variant thereof, as shown in SEQ ID NO.3; the second functional module includes a persistently activated cGAS mutant protein as shown in SEQ ID NO.1 or a lysosomal degradation-resistant derivative of the persistently activated cGAS mutant protein as shown in SEQ ID NO.

2. The N-terminal fragment of the cleavable intima-intima-peptide is fused to the C-terminus of the first functional module, and the C-terminal fragment of the cleavable intima-intima-peptide is fused to the N-terminus of the second functional module; wherein, the amino acid sequence of the N-terminal fragment of the cleavable intima-intima-peptide is as shown in SEQ ID NO.4, and the amino acid sequence of the C-terminal fragment of the cleavable intima-intima-peptide is as shown in SEQ ID NO.

5. The Cage-Colockr system is composed of the cage domain, Key domain and Bcl2 domain of amino acid sequences as shown in SEQ ID NO.6~SEQ ID NO.8, which are sequentially linked together. The cage domain is fused to the C-terminus of the first functional module and the Bcl2 domain is fused to the N-terminus of the second functional module.

2. The modular protein delivery system according to claim 1, characterized in that, The second functional module also includes an endoplasmic reticulum resident signal peptide KDEL sequence, which is fused to the C-terminus of the continuously activated cGAS mutant protein or its lysosomal degradation-resistant derivative. The amino acid sequence of the endoplasmic reticulum resident signal peptide KDEL is shown in SEQ ID NO.

9.

3. The modular protein delivery system according to claim 1 or 2, characterized in that, The first functional module further includes a targeting peptide fused to the N-terminus of the cholera exotoxin B subunit. The targeting peptide includes the M2pep targeting peptide, the amino acid sequence of which is shown in SEQ ID NO.

29.

4. The modular protein delivery system according to any one of claims 1-3, characterized in that, In the Cage-Colockr system, the Key domain is used to fuse a targeting ligand, which is a single-chain variable fragment antibody targeting PD-L1. The Key domain is fused with the single-chain variable fragment antibody targeting PD-L1 to form the Key-scFv modular protein delivery system. The amino acid sequence of the single-chain variable fragment antibody targeting PD-L1 is shown in SEQ ID NO.

10.

5. The modular protein delivery system according to any one of claims 1-4, characterized in that, The modular protein delivery system also includes an epitope tag, which is fused to the C-terminus of the second functional module; The epitope tag is selected from HA tag, Flag tag, His6 tag, c-Myc tag or V5 tag.

6. A gene encoding the modular protein delivery system according to any one of claims 1 to 5.

7. An expression carrier, characterized in that, It contains the gene as described in claim 6.

8. A host cell, characterized in that, The host cell contains the gene of claim 6 or the modular protein delivery system of any one of claims 1 to 5.

9. The use of the modular protein delivery system according to any one of claims 1 to 5, the gene according to claim 6, the expression vector according to claim 7, or the host cell according to claim 8 in the preparation of cell biology tools for anti-tumor immune research.

10. The use of the modular protein delivery system according to any one of claims 1 to 5, the gene according to claim 6, the expression vector according to claim 7, or the host cell according to claim 8 in the preparation of a medicament for the prevention or treatment of tumors, characterized in that, The tumor is selected from any one or more of melanoma, non-small cell lung cancer, breast cancer, colorectal cancer, liver cancer, breast cancer, and pancreatic tumor.

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