Bacteria-based protein delivery

Abstract

The present invention relates to recombinant Gram-negative bacterial strains and their use in methods of treating cancer in a subject.

Description

[Technical Field]

[0001] This invention relates to recombinant Gram-negative bacterial strains and their use in methods for treating target cancers. [Background technology]

[0002] Bacteria have evolved different mechanisms for directly injecting proteins into target cells. 1 Type III secretion apparatus (T3SS) used by bacteria such as Yersinia, Sigella, and Salmonella. 2 It functions like a nanosyringe for injecting so-called bacterial effector proteins into host cells. T3SS has been used to deliver hybrid peptides and proteins to target cells. Heteromicrobial T3SS effectors have been delivered when the bacteria under study are genetically largely unavailable (e.g., Chlamydia trachomatis). Often, reporter proteins have been fused with possible T3SS secretory signals for research requirements for T3SS-dependent protein delivery, such as Bordetella pertussis adenylyl cyclase, mouse DHFR, or phosphorylated tags. Peptide delivery has been primarily performed for vaccination purposes. This includes peptides representing viral epitopes, bacterial epitopes (Listerioridine O), and epitopes of human cancer cells. In a few cases, functional eukaryotic proteins have been delivered to modulate host cells, and these are nanobodies. 3 nucleoprotein (Cre-recombinase, MyoD) 4,5 Or Il10 and IL1ra 6 This is done using [specific method / technology]. None of the systems described above enable single-protein delivery, as each still encodes one or more endogenous effector proteins. Furthermore, the vectors used are not designed to allow for the simple cloning of other DNA fragments encoding the selected protein, hindering the widespread use of the system.

[0003] Approaches that enable targeted drug delivery are of great interest. For example, antibodies that recognize the surface structure of tumor cells and, in optimal cases, selectively bind to tumor cells are used. To improve the mechanism of such antibodies, they can be conjugated into lipid vesicles filled with therapeutic agents or drugs. One challenge with such vesicles is the proper release of the active reagent. Even more complex is the delivery of therapeutic proteins or peptides, especially when intracellular mechanisms are targeted. Many alternative methods have been attempted to solve the challenge of delivering therapeutic proteins into eukaryotic cells, including "cell-permeable peptides" (CPPs) or similar technologies, as well as various nanoparticle-based methods. All of these technologies have drawbacks, such as low efficacy and the high likelihood that the cargo taken up by cells via endocytosis will ultimately be degraded in lysosomes. Furthermore, the conflict between the need for stability of the cargo carrier in the human body and the need for destabilization and release within target cells constitutes an essential challenge for such technologies. Various bacteria, including Escherichia coli, Vibrio cholerae, Salmonella enterica, Listeria monocytogenes, Pseudomonas aeruginosa, and Bifidobacteria, have been shown to replicate within malignant solid tumors when administered distally. Currently, only Calmette-Guérin bacillus (BCG, derived from Mycobacterium bovis) is used in clinical practice. BCG is administered to treat superficial bladder cancer, but the underlying molecular mechanisms remain largely unknown. For example, developing strains that can deliver cargo produced inside the bacterium to its site of action inside cells, such as cancer cells—that is, outside the bacterium—remains a major challenge. [Overview of the project]

[0004] The present invention relates to recombinant Gram-negative bacterial strains and their use in methods for treating target cancers. In some embodiments, the present invention provides recombinant Gram-negative bacterial strains and their use for treating target cancers, wherein the recombinant Gram-negative bacterial strains enable the transfer of viral proteins, most importantly, various type III as well as type IV effectors of functional eukaryotic proteins, into cancer cells, such as malignant solid tumor cells.

[0005] The present invention provides a recombinant Gram-negative bacterial strain capable of expressing and secreting at least two different heterologous proteins, each at high peak levels, where, remarkably, the maximal activity of each heterologous protein is retained after delivery to eukaryotic cells, such as cancer cells, while maintaining the strain's complete tumor colonization ability and optimized genetic stability.

[0006] In the first aspect, the present invention is i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. This relates to a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules, wherein the first and second polynucleotide molecules are located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules are located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, provided that the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located.

[0007] In a further embodiment, the present invention is for use as a pharmaceutical, i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. This relates to a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules, wherein the first and second polynucleotide molecules are located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules are located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, provided that the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located.

[0008] In a further embodiment, the present invention relates to a method for treating a target cancer, comprising administering a recombinant Gram-negative bacterial strain to the target, wherein the recombinant Gram-negative bacterial strain is administered in an amount sufficient to treat the target, for use in such a method. i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. This relates to a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules, wherein the first and second polynucleotide molecules are located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules are located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, provided that the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located.

[0009] Similarly, the present invention relates to a method for treating a target cancer, comprising administering the recombinant Gram-negative bacterial strain described above to the target, wherein the recombinant Gram-negative bacterial strain is administered in an amount sufficient to treat the target.

[0010] Similarly, the present invention relates to the use of the above-mentioned recombinant Gram-negative bacterial strains in the manufacture of pharmaceuticals for treating target cancers. [Brief explanation of the drawing]

[0011] [Figure 1] Yersinia enterocolitica W227 pathogenic plasmid, pYV. A 69'673bp Yersinia pathogenic plasmid (pYV) of strain W227, drawn to a constant scale. T3SS effector protein, origin of replication, and arsenic resistance (encoded by genes arsC, B, R, and H) are shown: I: origin of replication, II: yopO, III: yopP, IV: yopQ, V: yopT, VI: sycT, VII: yopM, VIII: yopD, IX: yopB, X: sycD, XII: yopH, XIII: sycH, XIV: sycE, XV: yopE, XVI: yadA, XVII-XX: arsC, B, R, and H. [Figure 2]Modified Yersinia enterocolitica W227 pathogenic plasmid, pYV-051, encoding YopE1-138-human cGAS161-522 and YopE1-138-human RIG-I CARD2, respectively, on the endogenous sites of yopH and yopE of the endogenous pYV plasmid. 73,073 bp pYV-051 drawn at a constant scale. I: Replication origin (53...203), II: Destroyed YopO (7409...8116), III: Destroyed YopP (8597...8949), IV: YopQ (10692...11240), V: Destroyed YopT (11761...12301), VI: sycT (12301...12693), VII: Destroyed YopM (16270...17375), VIII: YopD (18629...19549), IX: YopB (19568...20773), X: sycD (20751...21257), XI: YopE1-138 (codon modification) (45228...4 5641); 50017…50754), XVII:yadA(60173…61636), XVIII:arsC(65122…65547), XIX:arsB(65560…66849), XX:arsR(66861…67215), XXI:arsH(67301…67999). [Figure 3] Description of vector pBad_Si2. (A) Vector map of the cloning plasmid pBad_Si2 used to construct a fusion construct with YopE1-138. The chaperone SycE and YopE1-138 fusion are located beneath the natural Y. enterocolitica promoter. (B) Multiple cloning site immediately following the yopE1-138 fragment on the pBad_Si2 plasmid. [Figure 4]Description of vector pT3P-715. Vector map of the medium copy number cloning plasmid pT3P-715 used to construct a fusion construct with YopE1-138. The chaperone SycE and YopE1-138 fusion are located under the natural Y. enterocolitica promoter. I: araBAD promoter region (4...279), II: PBAD promoter (250...277), III: MCSI (317...331), IV: SycE (339...731), V: YopE1-138 (924...1337), VI: MCS II (1338...1361), VII: c-Myc tag (1368...1397), VIII: 6His tag (1413...1430), IX: Stop codon (1431...1433), X: Chloramphenicol resistance (2110...2766), XI: pBR322 origin (2924...3552). [Figure 5] Description of vector pT3P-716. Vector map of the high copy number cloning plasmid pT3P-716 used to construct a fusion construct with YopE1-138. The chaperone SycE and YopE1-138 fusion are located under the natural Y. enterocolitica promoter. I: araBAD promoter region (4...279), II: PBAD promoter (250...277), III: MCSI (317...331), IV: SycE (339...731), V: YopE1-138 (924...1337), VI: MCS II (1338...1361), VII: c-Myc tag (1368...1397), VIII: 6His tag (1413...1430), IX: Stop codon (1431...1433), X: Chloramphenicol resistance (2110...2766), XI: ColE1 origin (2924...3552). [Figure 6]Description of vector pT3P-717. Vector map of the low-copy-number cloning plasmid pT3P-717 used to construct a fusion construct with YopE1-138. The chaperone SycE and YopE1-138 fusion are located under the natural Y. enterocolitica promoter. I: araBAD promoter region (4...279), II: PBAD promoter (250...277), III: MCSI (317...331), IV: SycE (339...731), V: YopE1-138 (924...1337), VI: MCS II (1338...1361), VII: c-Myc tag (1368...1397), VIII: 6His tag (1413...1430), IX: Stop codon (1431...1433), X: Chloramphenicol resistance (2110...2766), XI: pBR322 origin (2924...3552), XII: Rop (4124...4348). [Figure 7] Description of vector pT3P-751 encoding YopE1-138-human cGAS161-522 and YopE1-138-human RIG-I CARD2. Vector map of medium copy number vector pT3P-751 encoding YopE1-138-human cGAS161-522 and YopE1-138-human RIG-I CARD2 in a single operon under the control of the yopE promoter. I: araBAD promoter region (4...279), II: PBAD promoter (250...277), III: MCS I (317...331), IV: SycE (339...731), V: YopE1-138 (924...1337), VI: Human Rig1-Card2 (1350...2087), VII: YopE1-138 (codon modification) (2101...2514), VIII: Human cGas161-522 (2527...3614), IX: c-Myc tag (3626...3655), X: 6His tag (3671...3688), XI: Chloramphenicol resistance (4368...5024), XII: pBR322 origin (5182...5810). [Figure 8]Delivery of type I IFN-inducing proteins encoded by vectors or endogenous pathogenic plasmids. B16F1 IFN reporter cells were infected with a control strain of Y. enterocolitica ΔyopHOPEMT that did not deliver cargo (III), or with IV:YopE1-138-human RIG-I CARD2 (RIG-I1-245), V:YopE1-138-human RIG-I CARD2 and YopE1-138-human cGAS161-522 on an endogenous pYV plasmid, or with VI:YopE1-138-human RIG-I CARD2 encoded on a medium copy number vector. Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 9] Delivery of type I IFN-inducing proteins encoded by vectors or endogenous pathogenic plasmids. RAW macrophage IFN reporter cells were infected with Y. enterocolitica ΔyopHOPEMT, a control strain that did not deliver cargo (III), or with a medium copy number vector encoding YopE1-138-human cGAS161-522 (IV), or with an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 (V). Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 10]Delivery of type I IFN-inducing proteins encoded by vectors or endogenous pathogenic plasmids. B16F1 melanocyte IFN reporter cells were infected with Y. enterocolitica ΔyopHOPEMT, a control strain that did not deliver cargo (III), or a medium copy number vector encoding YopE1-138-mouse RIG-I CARD2 (RIG-I1-246) (IV), or an endogenous pYV plasmid encoding YopE1-138-mouse RIG-I CARD2 (V), or a medium copy number vector and an endogenous pYV plasmid both encoding YopE1-138-mouse RIG-I CARD2 (VI). Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 11] Delivery of type I IFN-inducing proteins encoded by vectors or endogenous pathogenic plasmids. B16F1 melanocyte IFN reporter cells were infected with a control strain of Y. enterocolitica ΔyopHOPEMT that did not deliver cargo (III), or with an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 (RIG-I1-245) and additionally with a medium copy number vector encoding YopE1-138-human RIG-I CARD2 (IV), or with both the endogenous pYV plasmid and the additional medium copy number vector encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 (V). Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 12]Delivery of type I IFN-inducing proteins encoded by vectors or endogenous pathogenic plasmids. B16F1 melanocytes (A), RAW macrophages (B), or THP-1 macrophages (C) IFN reporter cells are delivered to a control strain of Y. enterocolitica ΔyopHOPEMT that does not deliver cargo (III), or an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 (RIG-I1-245) (IV), or an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2, with additionally encoding YopE1-138-human cGAS161-522 on a medium copy number vector (V), or an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 was further infected with a medium copy number vector encoding YopE1-138-human RIG-I CARD2 (VI), or with both the endogenous pYV plasmid and the vector encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 (VII). Bacterial titration was performed for each strain added to cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 13]Immobilization of tumors with strains encoding type I IFN-inducible proteins on vectors or endogenous pathogenic plasmids. Delivery of human cGAS161-522 and RIG-I CARD2 (RIG-I1-245) and vector copy numbers did not alter the bacterial load within solid tumors in a 4T1 breast cancer model. Mice carrying subcutaneous 4T1 mammary tumors were given either (II) a control strain of Y. enterocolitica ΔyopHOPEMT with 5 × 10⁶ CFUs that did not deliver cargo, or (III) a strain encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on an endogenous pYV plasmid, or (IV) a strain encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on both an endogenous pYV plasmid and a medium copy number vector, or (V) a strain encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on both an endogenous pYV plasmid and a high copy number vector, or (V) a strain encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on both an endogenous pYV plasmid and a low copy number vector. The substance encoding CARD2 (VI) was administered intravenously. Bacterial load was determined as colony-forming units (CFU / g) per gram of tumor (I). [Figure 14] Expression and secretion of type I IFN-inducible proteins encoded by low / medium / high copy number vectors. Expression and secretion of human RIG-I CARD2 (RIG-I1-245) in relation to the vector copy number. Intrabacterial expression (I) or secretion into the supernatant (II) was evaluated for Y. enterocolitica ΔyopHOPEMT, a control strain that does not deliver cargo (III), or strains encoding YopE1-138-human RIG-I CARD2 on medium copy number vectors (IV), or strains encoding YopE1-138-human RIG-I CARD2 on high copy number vectors (V), or strains encoding YopE1-138-human RIG-I CARD2 on low copy number vectors (VI). [Figure 15]Delivery of endogenous pathogenic and type I IFN-inducing proteins encoded by low / medium / high copy number vectors. B16F1 IFN reporter cells were infected with a control strain of Y. enterocolitica ΔyopHOPEMT that did not deliver cargo (III), or with YopE1-138-human cGAS161-522 and human RIG-I CARD2 (RIG-I1-245) on an endogenous pYV plasmid, and additionally with a medium (IV), high (V), or low (VI) copy number vector encoding YopE1-138-human RIG-I CARD2. Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 16] Delivery of type I IFN-inducing proteins encoded by endogenous pathogenic plasmids and low / medium / high copy number vectors. B16F1 melanocytes (A), RAW macrophages (B), or THP-1 macrophages (C) IFN reporter cells were infected with a control strain of Y. enterocolitica ΔyopHOPEMT that does not deliver cargo (III), or with an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 (RIG-I1-245) (IV), or with an endogenous pYV plasmid encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2, with additional medium (V), high (VI), or low (VII) copy number vectors encoding YopE1-138-human cGAS161-522 and RIG-I CARD2. Bacterial titration was performed on each strain after adding it to cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured as optical density at 650 nm (II). [Figure 17]A strain for optimal delivery of endogenous pathogenic and vector-encoded type I IFN-inducible proteins. This concerns the delivery of YopE1-138-human cGAS161-522 and YopE1-138-RIG-I CARD2 (RIG-I1-245), encoded on the endogenous sites of yopH and yopE respectively in an endogenous pYV plasmid, and additionally on a medium copy number vector, where YopE1-138-human cGAS161-522 and YopE1-138-RIG-I CARD2 are encoded in a single operon under the control of the yopE promoter. [Figure 18] Delivery of type I IFN-inducing proteins cGAS and RIG-I CARD2 and their effects on different cell types. Delivery of human cGAS161-522 and RIG-I CARD2 (RIG-I1-245) results in differential induction of type I IFN signaling in B16F1 melanocytes (A), human glioblastoma LN-229 (B), mouse RAW macrophages (C), or human THP-1 macrophages (D). B16F1, RAW, or THP-1 IFN reporter cells and human LN-229 glioblastoma cells were infected with Y. enterocolitica ΔyopHOPEMT, a control strain that did not deliver cargo (III), or YopE1-138 encoding mouse RIG-I CARD2 on a medium copy number vector (IV), or YopE1-138 encoding human cGAS161-522 on a medium copy number vector (V). Bacterial titration was performed on each strain after adding it to cells, and the multiple of infections (MOI) is shown as I. Type I IFN induction was measured as optical density (II) at 650 nm for A, C, and D, and as pg / ml by IFNb ELISA for B. [Figure 19]Delivery of type I IFN-inducing proteins encoded by endogenous pathogenic plasmids and medium copy number vectors. Delivery of human cGAS161-522 and RIG-I CARD2 (RIG-I1-245) results in differential induction of type I IFN signaling in A20 B-cell lymphoma (A) or Jurkat T lymphocyte (B) cells. A) Mouse A20 lymphoma cells were infected with Y. enterocolitica ΔyopHOPEMT, a control strain that does not deliver cargo (III), or a medium copy number vector encoding YopE1-138-mouse RIG-I CARD2 (IV), or a medium copy number vector encoding YopE1-138-mouse RIG-I CARD2 and YopE1-138-human cGAS161-522 (V), or pYV and a medium copy number vector encoding YopE1-138-mouse RIG-I CARD2 and YopE1-138-human cGAS161-522 (VI). B) Human Jurkat T cells were infected with Y. enterocolitica ΔyopHOPEMT, a control strain that does not deliver cargo (III), or a medium copy number vector encoding YopE1-138-human RIG-I CARD2 and YopE1-138-human cGAS161-522 (IV), or pYV and a medium copy number vector encoding YopE1-138-human RIG-I CARD2 and YopE1-138-human cGAS161-522 (V). Bacterial titration was performed for each strain added to the cells, and the multiplicity of infection (MOI) is shown in I. Type I IFN induction was measured by ELISA with IFNb, and is shown as pg / ml for B (II). [Figure 20]Genetic stability of tumor-committed bacterial strains encoding type I IFN-inducible proteins on endogenous pathogenic plasmids. Encoding of human cGAS161-522 and RIG-I CARD2 (RIG-I1-245) is genetically stable when encoded in vivo on endogenous pYV pathogenic plasmids in a B16F10 melanoma model. Mice carrying subcutaneous B16F10 tumors were intravenously injected with 1 × 10⁶ CFU of Y. enterocolitica ΔyopHOPEMT encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on endogenous pYV plasmids. Bacteria were isolated 2 days after administration (animals 1 and 2) or 4 days after administration (animals 3-10), and replicas were collected on selective medium to evaluate the presence (I) or absence (II) of the pYV plasmid. [Figure 21] Genetic stability of tumor-complacent strains encoding type I IFN-inducible proteins on vectors and endogenous pathogenic plasmids. Encoding of human cGAS161-522 and RIG-I CARD2 (RIG-I1-245) on medium copy number vectors and endogenous pathogenic plasmids appears to be genetically more stable in vivo in the EMT-6 breast cancer model than encoding on endogenous pYV pathogenic plasmids or vectors alone. Mice carrying subcutaneous EMT-6 tumors were intratumorally injected with 7.5 × 10⁷ CFU of Y. enterocolitica ΔyopHOPEMT encoding YopE1-138-human cGAS161-522 and human RIG-I CARD2 on both endogenous pYV plasmids and medium copy number vectors. Bacteria were isolated on day 1 after administration (animals 1 and 2) or day 2 after administration (animals 4-6), and replicas were collected in selective medium to evaluate the presence of both the pYV plasmid and the medium copy vector (VI), the presence of only the pYV plasmid (V), the presence of only the medium copy vector (IV), or the absence of both the pYV plasmid and the medium copy vector (III). [Figure 22]Tumor progression in wild-type C57BL / 6 mice to which B16F10 melanoma cells were allografted subcutaneously. Wild-type C57BL / 6 mice, subcutaneously allografted with B16F10 melanoma cells, were intratumorally injected with either III:PBS, or 7.5 × 10⁷ CFU of IV: Y. enterocolitica dHOPEMT, or V: Y. enterocolitica dHOPEMT encoding YopE1-138-human cGAS161-522 and YopE1-138-human RIG-I CARD2 (RIG-I1-245). Both proteins are encoded in an endogenous pYV plasmid (containing the endogenous sites of yopH and yopE, respectively) and additionally in a medium copy number vector (on which YopE1-138-human cGAS161-522 and YopE1-138-RIG-I CARD2 are encoded in a single operon under the control of the yopE promoter). Intratumoral injection was initiated when the tumor reached a size of 61 (+ / -22) mm³. The day of the first intratumoral injection of bacteria was defined as day 0, and treatment was performed on d0, d1, d2, d3, d6, and d9. Tumor volume was measured with calipers over the following days (II: days). The mean tumor volume is shown in mm³ (I). [Figure 23] Tumor progression in wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells. Wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells were injected intratumor with PBS. Intratumor injection was started when the tumor reached a size of 61 (+ / -22) mm3. The day of the first intratumor injection was defined as day 0, and treatment was performed on d0, d1, d2, d3, d6, and d9. Tumor volume was measured with calipers over subsequent days (II: days). Tumor volume for individual animals (n=15) is shown in mm3 (I). [Figure 24]Tumor progression in wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells. Wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells were intratumorally injected with 7.5 × 10⁷ CFU of Y. enterocolitica dHOPEMT. Intratumoral injection was started when the tumor reached a size of 61 (+ / -22) mm³. The day of the first intratumoral injection was defined as day 0, and treatment was performed on d0, d1, d2, d3, d6, and d9. Tumor volume was measured with calipers over subsequent days (II: days). Tumor volume for individual animals (n=15) is shown in mm³ (I). [Figure 25] Tumor progression in wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells. Wild-type C57BL / 6 mice allografted subcutaneously with B16F10 melanoma cells were injected intratumor with 7.5 × 10⁷ CFU of Y. enterocolitica dHOPEMT encoding YopE1-138-human cGAS161-522 and YopE1-138-human RIG-I CARD2 (RIG-I1-245). Both proteins are encoded in an endogenous pYV plasmid (containing the endogenous sites of yopH and yopE, respectively) and additionally in a medium copy number vector (on which YopE1-138-human cGAS161-522 and YopE1-138-RIG-I CARD2 are encoded in a single operon under the control of the yopE promoter). Intratumoral injection was initiated when the tumor reached a size of 61 (+ / -22) mm³. The day of the first intratumoral injection was defined as day 0, and treatment was performed on d0, d1, d2, d3, d6, and d9. Tumor volume was measured with calipers over the following days (II: days). The tumor volume of individual animals (n=15) is shown in mm³ (I). [Figure 26]Delivery of type I interferon response-inducing proteins via bacterial T3SS-RIG1. Delivery of human and mouse RIG1 CARD domain variants results in type I IFN induction in RAW IFN reporter cell lines. RAW reporter cells were infected with Y. enterocolitica ΔHOPEMT encoding I:Y. enterocolitica ΔHOPEMT or pBad_Si2-derived plasmids, II:YopE1-138-human RIG1 CARD domains 1-245, III:YopE1-138-mouse RIG1 CARD domains 1-246, IV:YopE1-138-mouse RIG1 CARD domains 1-229, and V:YopE1-138-mouse RIG1 CARD domains 1-218. Bacterial titration (indicated as VI:MOI) was performed on each strain after adding bacteria to cells, and IFN stimulation was evaluated based on the activity of secreted alkaline phosphatase (VII:OD650) under the control of the I-ISG54 promoter, which consists of an IFN-inducible ISG54 promoter enhanced by the multimer ISRE. [Figure 27A-E] A list of strains used in this application. [Modes for carrying out the invention]

[0012] The present invention relates to recombinant Gram-negative bacterial strains and their use in methods for treating target cancers, such as malignant solid tumors.

[0013] For the purposes of interpreting this specification, the following definitions apply, and where appropriate, terms used in the singular also include their plural forms, and vice versa. It should be understood that the terminology used herein is solely for the purpose of describing specific embodiments and is not intended to be limiting.

[0014] The term "Gram-negative strain" used here includes the following bacteria: Aeromonas salmonicida, Aeromonas hydrophila, Aeromonas veronii, Anaeromyxobacter dehalogenans, Bordetella bronchiseptica, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia cepacia, and Glanders (Burkholderia cepacia). Burkholderia pseudomallei, Chlamydia muridarum, Chlamydia trachomatis, Chlamydophila abortus, Chlamydophila pneumoniae, Chromobacterium violaceum, Citrobacter rodentium, Desulfovibrio vulgaris, Edwardsiella tarda, Endozoicomonas elysicola, Erwinia amylovora, Escherichia albertii), Escherichia coli, Lawsonia intracellularis, Mesorhizobium rotii* Myxococcus xanthus, Pantoea agglomerans, Photobacterium damselae, Photorhabdus luminescens, Photorhabdus temperate, Pseudoalteromonas spongiae, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, Pseudomonas syringae, Ralstonia solanacearum, Rhizobium species, Salmonella Salmonella enterica and other Salmonella species, Shigella flexneri and other Shigella species, Sodalis glossinidius, Vibrio alginolyticus, Vibrio azureus, Vibrio campellii, Vibrio caribbenthicus, Vibrio harvey, Vibrio parahaemolyticus, Vibrio tasmaniensis, Vibrio tubiashii, canker bacterium (Xanthomonas axonopodis), Xanthomonas campestris Campestris), rice bacterial leaf blight fungus (Xanthomonas oryzae), Enterocolitica bacteria (Yersinia enterocolitica), plague bacterium (Yersinia pestis), pseudotuberculosis bacterium (Yersinia(pseudotuberculosis). Preferred Gram-negative bacterial strains of the present invention are Gram-negative bacterial strains belonging to the Enterobacteriaceae and Pseudomonasaceae families. Gram-negative bacterial strains of the present invention are typically used in vitro and / or in vivo, preferably in vivo, to deliver heterologous proteins to eukaryotic cells by bacterial T3SS.

[0015] The term "recombinant Gram-negative strain" as used herein refers to a recombinant Gram-negative strain that has been genetically transformed with a vector-like polynucleotide construct. The pathogenicity of such recombinant Gram-negative strains is typically attenuated by the absence of a pathogenic bacterial effector protein, which is transported by one or more bacterial proteins that are part of the secretory system. Such effector proteins are delivered into host cells by the secretory system, where they exert their pathogenic activity against various host proteins and cellular mechanisms. Many different effector proteins are known, transported by various types of secretory systems, and exhibit a broad repertoire of biochemical activities that modulate the function of host regulatory molecules. The pathogenicity of the recombinant Gram-negative strains used herein may be further attenuated by the absence of siderophores, which are normally or occasionally produced by Gram-negative strains; therefore, the strain does not produce siderophores, for example, it is deficient in siderophore production. Accordingly, in a preferred embodiment, a recombinant Gram-negative bacterial strain lacking siderophores, which are normally or occasionally produced by Gram-negative bacterial strains, is used. Therefore, the strain does not produce siderophores, for example, and is deficient in siderophore production. More preferably, a Yersinia strain lacking siderophores, which are normally or occasionally produced by Gram-negative bacterial strains, is used, particularly Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T, Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T ΔHairpinI-virF, or Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T Δasd pYV-asd. Therefore, the strain does not produce siderophores, for example, and is deficient in siderophore production, particularly in yersinia bactin production. Most preferably, Yersinia strains lacking siderophores, which are normally or occasionally produced by Gram-negative bacterial strains, particularly Y. enterocolitica MRS40 ΔyopH, O, P, E, M, T, are used, and so the strain does not produce siderophores, for example, it is deficient in siderophore production, and in particular, it is deficient in Yersinia bactin production.Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T, which lacks yersinia bactin production, is described in International Publication No. 02077249 and was deposited with the Belgian Microbial Coordinated Collection (BCCM) on September 24, 2001, in accordance with the Budapest Convention for the International Recognition of Deposit of Microorganisms in Patent Proceedings, and was assigned accession number LMG P-21013. Recombinant Gram-negative strains preferably do not produce siderophores, for example, are deficient in siderophore production.

[0016] The terms "siderofore," "iron siderofore," or "iron chelating agent," used interchangeably here, refer to compounds that have a high affinity for iron, such as small compounds that have a high affinity for iron.

[0017] Gram-negative siderophores include, for example, enterobactin and dihydroxybenzoylserine synthesized by the genera Salmonella, Escherichia coli, Sigella, and Serratia (but used by all intestinal bacteria); pioverdin synthesized by Pseudomonas; vibriobactin synthesized by Vibrio; acinetobactin and acinetoferrin synthesized by Acinetobacter; yersinia bactin and aerobactin synthesized by Yersinia; ornibactin synthesized by Burkholderia; salmonokerin synthesized by Salmonella; aerobactin synthesized by Escherichia coli, Sigella, Salmonella, and Yersinia; alkalidin synthesized by Bordetella; and viscaberin synthesized by Vibrio.

[0018] Siderophores include hydroxamates, catecholates, and mixed ligand siderophores. Several siderophores have been approved for human use to date, primarily for the treatment of iron overload. Preferred siderophores are deferoxamine (also known as desferrioxamine B, desferoxamine B, DFO-B, DFOA, DFB, or desferal), desferrioxamine E, deferasirox (Exjade, Desirox, Defriget, Decipher), and deferipron (Ferriprox).

[0019] The term "endogenous protein essential for growth" used here refers to proteins in recombinant Gram-negative bacterial strains that cannot grow without them. Endogenous proteins essential for growth include, for example, enzymes essential for amino acid production, enzymes involved in peptidoglycan biosynthesis, enzymes involved in LPS biosynthesis, enzymes involved in nucleotide synthesis, or translation initiation factors.

[0020] The term "enzymes essential for amino acid production" used here refers to enzymes involved in amino acid production in recombinant Gram-negative bacterial strains, without which the Gram-negative strains cannot grow. Examples of enzymes essential for amino acid production include aspartate-β-semialdehyde dehydrogenase (asd), glutamine synthase (glnA), tryptophanyl-tRNA synthase (trpS), or serine hydroxymethyltransferase (glyA), or transketolase 1 (tktA), transketolase 2 (tktB), ribulose-phosphate 3-epimerase (rpe), ribose-5-phosphate isomerase A (rpiA), transaldolase A (talA), and transaldolase. B(talB), phosphoribosyl pyrophosphate synthase (prs), ATP phosphoribosyltransferase (hisG), histidine biosynthesis bifunctional protein HisIE (hisI), 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylideneamino]imidazole-4-carboxamide isomerase (hisA), imidazole glycerol phosphate synthase subunit HisH (hisH), imidazole glycerol phosphate synthase subunit HisF (hisF) Histidine biosynthesis bifunctional protein HisB (hisB), histidinol-phosphate aminotransferase (hisC), histidinol dehydrogenase (hisD), 3-dehydroquinate synthase (aroB), 3-dehydroquinate dehydratase (aroD), shikimate dehydrogenase (NADP(+)) (aroE), shikimate kinase 2 (aroL), shikimate kinase 1 (aroK), 3-phosphoshikimate 1-carboxyvinyltransferase (aroA), chorismylate synthase Tase (aroC), P-protein (pheA), T-protein (tyrA), aromatic amino acid aminotransferase (tyrB), phospho-2-dehydro-3-deoxyheptone aldolase (aroG), phospho-2-dehydro-3-deoxyheptone aldolase (aroH), phospho-2-dehydro-3-deoxyheptone aldolase (aroF), quinic acid / shikimic acid dehydrogenase (ydiB), ATP-dependent 6-phosphofructokinase isozyme 1 (pfkA),ATP-dependent 6-phosphofructokinase isozyme 2 (pfkB), fructose-diphosphate aldolase class 2 (fbaA), fructose-diphosphate aldolase class 1 (fbaB), triose phosphate isomerase (tpiA), pyruvate kinase I (pykF), pyruvate kinase II (pykA), glyceraldehyde-3-phosphate dehydrogenase A (gapA), phosphoglycerate kinase (pgk), 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA), 2,3-bisphosphoglycerate Phosphate-independent phosphoglycerate mutase (gpmM / yibO), putative phosphoglycerate mutase (ytjC / gpmB), enolase (eno), D-3-phosphoglycerate dehydrogenase (serA), phosphoserine aminotransferase (serC), phosphoserine phosphatase (serB), L-serine dehydratase 1 (sdaA), L-serine dehydratase 2 (sdaB), L-threonine dehydratase catabolism (tdcB), L-threonine dehydratase biosynthesis (ilvA), L-serine dehydratase (td cG), serine acetyltransferase (cysE), cysteine ​​synthase A (cysK), cysteine ​​synthase B (cysM), beta-cystathionase (malY), cystathionine beta-lyase (metC), 5-methyltetrahydropteroyltriglutamate-homocysteine ​​methyltransferase (metE), methionine synthase (metH), S-adenosylmethionine synthase (metK), cystathionine gamma-synthase (metB), homoserine O-succinyltransferase (metA ), 5'-methylthioadenosine / S-adenosylhomocysteine ​​nucleosidase (mtnN), S-ribosylhomocysteine ​​lyase (luxS), cystathione beta-lyase, cystathione gamma-lyase, serine hydroxymethyltransferase (glyA), glycine hydroxymethyltransferase (itaE), 3-isopropylmalate dehydratase small subunit (leuD), 3-isopropylmalate dehydratase large subunit (leuC), 3-isopropylmalate dehydrogenase (leuB),L-threonine dehydratase biosynthesis (ilvA), acetolactic acid synthase isozyme 3 major subunits (ilvI), acetolactic acid synthase isozyme 3 minor subunits (ilvH), acetolactic acid synthase isozyme 1 minor subunit (ilvN), acetolactic acid synthase isozyme 2 minor subunits (ilvM), ketolate reductosomerase (NADP(+)) (ilvC), dihydroxy acid dehydratase (ilvD), branched-chain amino acid aminotransferase (ilvE), bifunctional aspart kinase / homoserine dehydrase Rogenase 1 (thrA), bifunctional aspart kinase / homoserine dehydrogenase 2 (metL), 2-isopropylmalate synthase (leuA), glutamate-pyruvate aminotransferase (alaA), aspartate aminotransferase (aspC), bifunctional aspart kinase / homoserine dehydrogenase 1 (thrA), bifunctional aspart kinase / homoserine dehydrogenase 2 (metL), lysine-sensitive aspart kinase 3 (lysC), aspartate-semialdehyde dehydrogenase (as d) 2-keto-3-deoxy-galactonate aldolase (yagE), 4-hydroxytetrahydrodipicolinate synthase (dapA), 4-hydroxytetrahydrodipicolinate reductase (dapB), 2,3,4,5-tetrahydropyridine-2,6-dicarboxylate N-succinyltransferase (dapD), succinyl-diaminopimerate desuccinylase (dapE), diaminopimerate epimerase (dapF), putative lyase (yjhH), acetylornithine / succinyldiaminopimerate aminotransferase Aconitate hydratase (argD), citrate synthase (gltA), aconitate hydratase B (acnB), aconitate hydratase A (acnA), uncharacterized putative aconitate hydratase (ybhJ), isocitrate dehydrogenase (icd), aspartate aminotransferase (aspC), glutamate-pyruvate aminotransferase (alaA), glutamate synthase [NADPH] large chain (gltB), glutamate synthase [NADPH] small chain (gltD), glutamine synthase (glnA),Amino acid acetyltransferase (argA), acetylglutamate kinase (argB), N-acetyl-γ-glutamyl-phosphate reductase (argC), acetylornithine / succinyl diaminopimelate aminotransferase (argD), acetylornithine deacetylase (argE), ornithine carbamoyltransferase chain F (argF), ornithine carbamoyltransferase chain I (argI), argininosuccinate tRNA ligase (argG), argininosuccinate lyase (argH), glutamate 5-kinase (proB), γ-glutamyl phosphate reductase (proA), pyrroline-5-carboxylate reductase (proC), ornithine cyclodeaminase, leucine-tRNA ligase (leuS), glutamine-tRNA ligase (glnS), serine-tRNA ligase (serS), glycine-tRNA ligase β-subunit (glyS), glycine-tRNA ligase Gauze α subunit (glyQ), tyrosine-tRNA ligase (tyrS), threonine-tRNA ligase (thrS), phenylalanine-tRNA ligase α subunit (pheS), phenylalanine-tRNA ligase β subunit (pheT), arginine-tRNA ligase (argS), histidine-tRNA ligase (hisS), valine-tRNA ligase (valS), alanine-tRNA ligase (alaS), isoleucine-tRNA These include ligase (ileS), proline-tRNA ligase (proS), cysteine-tRNA ligase (cysS), asparagine-tRNA ligase (asnS), aspartic acid-tRNA ligase (aspS), glutamic acid-tRNA ligase (gltX), tryptophan-tRNA ligase (trpS), glycine-tRNA ligase β-subunit (glyS), methionine-tRNA ligase (metG), and lysine-tRNA ligase (lysS). The preferred enzymes essential for amino acid production are tktA, rpe, prs, aroK, tyrB, aroH, fbaA, gapA, pgk, eno, tdcG, cysE, metK, glyA, asd, dapA / B / D / E / F, argC, proC, leuS, glnS, serS, glyS / Q, tyrS, thrS, pheS / T,argS, hisS, valS, alaS, ileS, proS, cysS, asnS, aspS, gltX, trpS, glyS, metG, lysS are preferred, and more preferred are asd, glyA, leuS, glnS, serS, glyS / Q, tyrS, thrS, pheS / T, argS, hisS, valS, alaS, ileS, proS, cysS, asnS, aspS, gltX, trpS, glyS, metG, lysS, with the most preferred being asd.

[0021] The terms "Gram-negative bacterial strain lacking the production of an amino acid essential for growth" and "nutrient requirement mutant" are used interchangeably here and refer to a Gram-negative bacterial strain that cannot grow without at least one exogenously supplied essential amino acid or its precursor. Examples of amino acids lacking production in such strains include aspartic acid, meso-2,6-diaminopimelic acid, aromatic amino acids, or leucine-arginine. Such strains can be produced, for example, by a deletion of the aspartic acid-beta-semialdehyde dehydrogenase gene (Δasd). Such nutrient requirement mutants cannot grow without exogenous meso-2,6-diaminopimelic acid. In the present invention, for the Gram-negative bacterial strain lacking the production of an amino acid essential for growth, a mutation, such as a deletion of the aspartic acid-beta-semialdehyde dehydrogenase gene, is preferred.

[0022] The term "Gram-negative bacterial strain lacking the production of adhesion proteins that bind to the eukaryotic cell surface or extracellular matrix" refers to a mutant Gram-negative bacterial strain that does not express at least one adhesion protein compared to the adhesion proteins expressed by the corresponding wild-type strain. Adhesion proteins may include elongated polymer adhesion molecules such as pili / fimbriae or non-fimbriae adherens. Fimbriacid adherens include type 1 pili (e.g., E. coli Fim pili with FimH adheren), type P pili (e.g., Pap pili with PapG adheren from E. coli), type 4 pili (e.g., pyrin protein from Pseudomonas aeruginosa), or curli (Csg protein with CsgA adheren from Salmonella). Non-fimbritic adhesins may include trimer autotransporter adhesins such as YadA, BpaA (B. pseudomallei), Hia (Haemophilus influenzae), BadA (B. henselae), NadA (N. meningitidis), or UspA1 (M. catarrhalis) from Y. enterocolitica, as well as other autotransporter adhesins such as AIDA-1 (Escherichia coli), and other adhesins / invasins such as InvA or intimin (Escherichia coli) or members of the Dr family or Afa family (Escherichia coli) from Y. enterocolitica. The terms YadA and InvA used here refer to proteins derived from Y. enterocolitica. Autotransporter YadA 7 While it binds to different types of collagen and fibronectin, Invasin InvA 8 It binds to β-integrin in the eukaryotic cell membrane. If the Gram-negative bacterial strain is a Y. enterocolitica strain, it is preferable that the strain has a deficiency of InvA and / or YadA.

[0023] As used herein, the term "Enterobacteriaceae" encompasses a family of Gram-negative, rod-shaped, facultative anaerobic bacteria found in soil, water, plants, and animals, which frequently occur as pathogens in vertebrates. Bacteria of this family share similar physiological properties, and conservation of functional elements and genes within their respective genomes has been demonstrated. Not only are they oxidase-negative, but all members of this family are glucose fermenters, and most are nitrate reducers.

[0024] The Enterobacteriaceae bacteria of the present invention may be any bacteria of that family and may include, but are not limited to, bacteria of the following genera: Escherichia coli, Sigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Erwinia, Morganella, Providencia, or Yersinia. In more specific embodiments, the bacteria include Escherichia coli, Escherichia blattae, Escherichia fergusonii, Escherichia hermanii, Escherichia vuneris, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonei, Enterobacter aerogenes, Enterobacter gergoviae, and Enterobacter sakazakii. These are bacteria of the species *Sakazakii*, *Enterobacter cloacae*, *Enterobacter agglomerans*, *Klebsiella pneumoniae*, *Klebsiella oxytoca*, *Serratia marcescens*, *Mycobacterium pseudotuberculosis*, *Plague bacillus*, *Enterocolitica*, *Clostridium erythrorhizon*, *Proteus mirabilis*, *Proteus vulgaris*, *Proteus penneri*, *Proteus hauseri*, *Providencia alcalifaciens*, or *Morganella morganii*.

[0025] Preferably, the Gram-negative bacterial strain is selected from the group consisting of the genera Yersinia, Escherichia coli, Salmonella, Sigella, Pseudomonas, Chlamydia, Erwinia, Pantoea, Vibrio, Burkholderia, Ralstonia, Xanthomonas, Chromobacterium, Sodalis, Citrobacter, Edwardsiella, Rhizobiae, Aeromonas, Photorhabdus, Bordetella, and Desulfovibrio; more preferably from the group consisting of Yersinia, Escherichia coli, Salmonella, and Pseudomonas; most preferably from the group consisting of Yersinia and Salmonella; and particularly from the genus Yersinia.

[0026] The term "Yersinia" as used herein includes all species of the genus Yersinia, including Enterocolitica, Mycobacterium pseudotuberculosis, and Yersinia pestis. Enterocolitica is preferred.

[0027] The term "Salmonella" as used herein includes all species of the genus Salmonella, including Salmonella bacillus and Salmonella bongoli. Salmonella bacillus is preferred.

[0028] As used herein, "promoter" refers to a nucleic acid sequence that regulates the expression of a transcription unit. The "promoter region" is a regulatory region that binds to intracellular RNA polymerase and initiates the transcription of the downstream (3' direction) coding sequence. Within the promoter region, transcription initiation sites (concretely defined by mapping using nuclease S1), as well as protein-binding domains (consensus sequences) involved in RNA polymerase binding, such as the putative -35 region and the Pribno box, are found. The term "functionally linked" simply means that, when describing the relationship between two nucleotides, for example, DNA regions, they are functionally related to each other and located on the same nucleic acid fragment. A promoter is functionally linked to a structural gene if it controls the transcription of that gene and is located on the same nucleic acid fragment as the gene. Typically, the promoter is functional in the Gram-negative strain; that is, the promoter can express the fusion protein of the present invention; that is, the promoter can express the fusion protein of the present invention without further genetic modification or further protein expression. Furthermore, the functional promoter should not be spontaneously deregulated in the bacterium T3SS.

[0029] As used herein, the term “extrachromosomal genetic elements” refers to exogenous genetic elements that are endogenously held by the Gram-negative bacterial strains of the present invention, such as pathogenic plasmids, or that are transiently or stably incorporated into or into non-chromosomal genetic elements that are endogenously held, such as endogenous pathogenic plasmids, or into non-chromosomal genetic elements that are transformed with such elements. Endogenous pathogenic plasmids are preferred extrachromosomal genetic elements of the present invention. Such extrachromosomal genetic elements may be generated by the incorporation of a vector, such as an expression vector, homologous recombination, or a vector for other incorporation into or into a non-chromosomal genetic element that is endogenously held, such as a pathogenic plasmid; by the incorporation of a DNA fragment for other incorporation into or into a non-chromosomal genetic element that is endogenously held, such as a pathogenic plasmid; or by RNA element guide site-specific insertion into or into a non-chromosomal genetic element that is endogenously held, such as a pathogenic plasmid, such as CRISPR / Cas9 and associated guide RNA.

[0030] The terms "polynucleic acid molecule" and "polynucleotide molecule" are used interchangeably and have the same meaning here, referring to both DNA and RNA molecules that can be single-stranded or double-stranded and can be partially or completely transcribed and translated (DNA) or partially or completely translated (RNA) into gene products.

[0031] The terms “nucleic acid sequence,” “nucleotide sequence,” and “nucleotide acid sequence” are used interchangeably here and have the same meaning, preferably referring to DNA or RNA. The terms “nucleic acid sequence,” “nucleotide sequence,” and “nucleotide acid sequence” are preferably used synonymously with the term “polynucleotide sequence.”

[0032] The term "operon" used here refers to two or more genes transcribed under the control of a single promoter. Therefore, these genes are typically transcribed together to form a single messenger RNA, and this single mRNA encodes more than one protein (polycistronic mRNA). In addition to the promoter and the two or more genes, operator elements that regulate transcription may also be present.

[0033] As used herein, the term "delivery" refers to the transport of proteins from recombinant Gram-negative bacterial strains to eukaryotic cells, comprising the steps of expressing a heterologous protein in a recombinant Gram-negative bacterial strain, secreting the expressed protein from such recombinant Gram-negative bacterial strain, and transferring the protein secreted by such Gram-negative bacterial strain into the cytosol of eukaryotic cells. Accordingly, the terms "delivery signal" or "secretion signal," used interchangeably here, refer to polypeptide sequences that are recognizable by the secretory and translocation systems of Gram-negative bacterial strains and direct the delivery of proteins from Gram-negative bacterial strains to eukaryotic cells.

[0034] As used herein, the term “delivery signal from bacterial effector protein” refers to a delivery signal from a bacterial effector protein that is functional in recombinant Gram-negative strains, i.e., enables heterologous proteins expressed in recombinant Gram-negative strains to be secreted from such recombinant Gram-negative strains by secretion systems such as type III, type IV, or type VI secretion systems, or to be transferred by such recombinant Gram-negative strains into the cytosol of eukaryotic cells by such secretion systems such as type III, type IV, or type VI secretion systems. As used herein, the term “delivery signal from bacterial effector protein” also includes fragments of delivery signals from bacterial effector proteins, i.e., shorter forms of delivery signals, for example, delivery signals containing up to 10, preferably up to 20, more preferably up to 50, even more preferably up to 100, and particularly up to 140 amino acids of a naturally occurring delivery signal. Therefore, a nucleotide sequence, such as a DNA sequence, that codes for a delivery signal from a bacterial effector protein may code for a full-length delivery signal or a fragment thereof, where the fragment typically contains up to 30 nucleic acids, preferably up to 60, more preferably up to 150, even more preferably up to 300, and particularly up to 420.

[0035] In this context, "secretion" of proteins refers to the transport of heterologous proteins across the cell membrane of recombinant Gram-negative bacterial strains to the outside. "Transfer" of proteins refers to the transport of heterologous proteins from recombinant Gram-negative bacterial strains across the cell membrane of eukaryotic cells to the cytosol of such eukaryotic cells.

[0036] The term "bacterial proteins that are part of the secretory system" as used herein refers to bacterial proteins that constitute the essential components of the bacterial type 3 secretory system (T3SS), type 4 secretory system (T4SS), and type 6 secretory system (T6SS), preferably T3SS. Without such proteins, each secretory system would not function in the transfer of proteins to the host cell, even if all other components of the secretory system and the translocated bacterial effector proteins were still encoded and produced.

[0037] The term "bacterial effector protein" as used herein refers to bacterial proteins transported by secretory systems, such as bacterial proteins that are part of the secretory mechanism into host cells. Such effector proteins are delivered to host cells by secretory systems, where they exert, for example, pathogenic activity against various host proteins and cellular mechanisms. Many different effector proteins are known, transported by various types of secretory systems, and exhibit a broad repertoire of biochemical activities that modulate the function of host regulatory molecules. Secretory systems include type 3 secretory system (T3SS), type 4 secretory system (T4SS), and type 6 secretory system (T6SS). Some effector proteins (such as Sigella flexneri IpaC) also belong to the class of bacterial proteins, are part of the secretory mechanism, and enable protein translocation. The recombinant Gram-negative bacterial strains used herein typically contain bacterial proteins that constitute essential components of the bacterial type 3 secretory system (T3SS), type 4 secretory system (T4SS), and / or type 6 secretory system (T6SS), preferably type 3 secretory system (T3SS). The term "bacterial proteins constituting essential components of bacterial T3SS" as used herein refers to proteins that naturally form injectosomes, such as injection needles, or are otherwise essential for their function in the transfer of proteins into eukaryotic cells. Proteins that form injectosomes or are otherwise essential for their function in the transfer of proteins into eukaryotic cells include, but are not limited to, SctC, YscC, MxiD, InvG, SsaC, EscC, HrcC, HrcC (secretin), SctD, YscD, MxiG, Prg, SsaD, EscD, HrpQ, HrpW, FliG (outer MS ring protein), SctJ, YscJ, MxiJ, PrgK, SsaJ, EscJ, HrcJ, Hr cJ, FliF (inner MS ring protein), SctR, YscR, Spa24, SpaP, SpaP, SsaR, EscR, HrcR, HrcR, FliP (minor export machinery protein), SctS, YscS, Spa9 (SpaQ), SpaQ, SsaS, EscS, HrcS, HrcS, FliQ (minor export machinery protein), SctT, YscT, Spa29 (SpaR), SpaR, SsaT, EscT, HrcT, HrcT,FliR (minor export machinery protein), SctU, YscU, Spa40, SpaS, SpaS, SsaU, EscU, HrcU, HrcU, FlhB (export machinery switch protein), SctV, YscV, MxiA, InvA, SsaV, EscV, HrcV, HrcV, FlhA (major export machinery protein), SctK, YscK, MxiK, OrgA, HrpD (accessory cytoplasmic protein), SctQ, YscQ, Spa3 3, SpaO, SpaO, SsaQ, EscQ, HrcQA+B, HrcQ, FliM+FliN (C-ring protein), SctL, YscL, MxiN, OrgB, SsaK, EscL, Orf5, HrpE, HrpF, FliH (stater), SctN, YscN, Spa47, SpaL, InvC, SsaN, EscN, HrcN, HrcN, FliI (ATPase), SctO, YscO, Spa13, SpAM, Invi, SsaO, Orf15, HrpO, HrpD, FliJ (Stork), SctF, YscF, MxiH, PrgI, SsaG, EscF, HrpA, HrpY (Needle Filament Protein), SctI, YscI, MxiI, PrgJ, SsaI, EscI, rOrf8, HrpB, HrpJ (Inner Rod Protein), SctP, YscP, Spa32, SpaN, InvJ, SsaP, EscP, Orf16, HrpP, HpaP, FliK (Needle Length Regulator), LcrV, IpaD, Si This includes pD (hydrophilic translocator, needle tip protein), YopB, IpaB, SipB, SseC, EspD, HrpK, PopF1, PopF2 (hydrophobic translocator, pore protein), YopD, IpaC, SipC, SseD, EspB (hydrophobic translocator, pore protein), YscW, MxiM, InvH (Pilotin), SctW, YopN, MxiC, InvE, SsaL, SepL, HrpJ, and HpaA (gatekeeper).

[0038] The terms "T6SS effector protein" or "bacterial T6SS effector protein" as used herein refer to proteins spontaneously injected into the cytosol of eukaryotic cells or into bacteria by the T6S apparatus, and proteins spontaneously secreted by the T6S apparatus that may, for example, form transition pores into the eukaryotic membrane. The terms "T4SS effector protein" or "bacterial T4SS effector protein" as used herein refer to proteins spontaneously injected into the cytosol of eukaryotic cells by the T4S apparatus, and proteins spontaneously secreted by the T4S apparatus that may, for example, form transition pores into the eukaryotic membrane.

[0039] The terms “T3SS effector protein” or “bacterial T3SS effector protein” as used herein refer to proteins spontaneously injected into the cytosol of eukaryotic cells by the T3S apparatus (including pore-forming translocators (such as Yersinia YopB and YopD) and tip proteins such as Yersinia LcrV), and proteins spontaneously secreted by the T3S apparatus that may, for example, form transition pores into the eukaryotic membrane. Preferably, proteins spontaneously injected into the cytosol of eukaryotic cells by the T3S apparatus are used. These pathogenic factors paralyze or reprogram eukaryotic cells to favor the pathogen.T3S effectors exhibit a broad repertoire of biochemical activity, regulating the function of important host regulatory molecules, including but not limited to AvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, and CigR , EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA protein family, HopAB2, HopAO1, HopI1, HopM1, Ho pN1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopU1, HsvB, IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8, IpaH9.8, I pgB1, IpgB2, IpgD, LcrV, Map, OspC1, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXo1, PthXo6, P thXo7, SifA, SifB, SipA / SspA, SipB, SipC / SspC, SipD / SspD, SlrP, SopA, SopB / SigD, SopD, SopE, SopE2, Sp iC / SsaB, SptP, SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC, SseD, SseF, SseG, SseI / SrfH, SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA, SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB, YopD Including YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA.

[0040] As used herein, the term "recombinant Gram-negative strain that accumulates in malignant solid tumors" or "the recombinant Gram-negative strain accumulates in malignant solid tumors" refers to a recombinant Gram-negative strain that replicates within malignant solid tumors and increases the bacterial count of said recombinant Gram-negative strain within the malignant solid tumor. Surprisingly, it has been found that after administration to a subject, the recombinant Gram-negative strain specifically accumulates in malignant solid tumors, that is, specifically accumulates in organs where malignant solid tumors are present, and in organs where no malignant solid tumor is present, the bacterial count of the recombinant Gram-negative strain is low or undetectable.

[0041] In the case of extracellular bacteria such as those of the genus Yersinia, the bacteria mainly accumulate in the intercellular spaces formed between tumor cells or between cells of the tumor microenvironment. Bacteria that proliferate intracellularly, such as those of the genus Salmonella, mainly invade tumor cells or cells of the tumor microenvironment and reside within such cells, although extracellular accumulation may still occur. The bacterial count of the recombinant Gram-negative strain accumulated in a malignant solid tumor is, for example, 10 per gram of tumor tissue 4 to 10 9 bacteria, which may be within the range of

[0042] As used herein, the term "cancer" refers to a disease in which abnormal cells divide uncontrollably and can invade adjacent tissues. Cancer cells can also spread to other parts of the body via the blood and lymphatic system. There are several main types of cancer. Carcinoma is cancer that arises in the tissue that lines or covers the skin or internal organs. Sarcoma is cancer that arises in bone, cartilage, fat, muscle, blood vessels, or other connective or supporting tissue. Leukemia is cancer that arises in hematopoietic tissues such as bone marrow, produces a large number of abnormal blood cells that infiltrate the bloodstream. Lymphoma and multiple myeloma are cancers that arise in cells of the immune system. Central nervous system cancer is cancer that arises in tissues of the brain and spinal cord. As used herein, the term "cancer" includes solid tumors, namely malignant solid tumors such as sarcoma, carcinoma, and lymphoma, as well as non-solid tumors such as leukemia (blood cancer). Malignant solid tumors are preferred.

[0043] The terms “solid tumor,” “solid tumor symptom,” “malignant solid tumor,” or “malignant solid tumor symptom” as used herein refer to an abnormal mass of tissue that does not typically contain a cyst or fluid area. Solid tumors can be benign (not cancerous) or malignant (cancerous). Malignant solid tumors are treated by the methods of the present invention. Different types of malignant solid tumors are named after the cell type that forms them. Examples of malignant solid tumors are sarcomas, carcinomas, and lymphomas. Leukemia (cancer of the blood) does not generally form malignant solid tumors (as defined by the National Cancer Institute of the NIH). Malignant solid tumors include, but are not limited to, abnormal cell masses that may arise from different tissue types such as the liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lips, oral cavity, esophagus, ovaries, prostate, stomach, testes, thyroid, or lung, and therefore include malignant solid liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lips, oral cavity, esophagus, ovaries, prostate, stomach, testes, thyroid, or lung tumors. Preferred malignant solid tumors that can be treated by the method of the present invention are malignant solid tumors arising from the skin, breast, liver, pancreas, bladder, prostate, and colon, and therefore include malignant solid skin, breast, liver, pancreas, bladder, prostate, and colon tumors. Equally preferred malignant solid tumors that can be treated by the method of the present invention are malignant solid tumors associated with liver cancer, such as hepatocellular carcinoma.

[0044] The term "objective response rate" (ORR) used here refers to the percentage of patients who experience a predetermined reduction in tumor size over a minimum period. The duration of response is typically measured from the initial response until tumor progression is observed. Generally, the FDA defines ORR as the sum of partial and complete responses. When defined in this way, ORR becomes a direct measure of drug antitumor activity and can be evaluated in single-arm trials. ORR refers to the sum of complete responses (CR) and partial responses (PR). The definitions of human ORR, CR, and PR are given in the RECIST guidelines (RECIST 1.1). 69 ) and conformity guidelines for the evaluation of immunotherapy compounds (iRECIST) 70 It is provided to ).

[0045] In preclinical studies using tumor-bearing mice, the definition of tumor response has been adapted to the human RECIST definition: no tumor regression is defined as an increase of more than 35% in tumor volume compared to each volume on day 0; stable disease is defined as a tumor volume change between a 50% decrease and a 35% increase compared to day 0; partial regression is defined as a tumor volume reduction between 50% and 95% compared to day 0; and complete regression or complete response is defined as a tumor volume reduction of >95% compared to day 0.

[0046] The terms "complete response," "complete tumor regression," and "complete regression" are used interchangeably and have the same meaning here. The term "complete response" (CR) in relation to target lesions refers to the disappearance of all target lesions. Pathogenic lymph nodes (whether target or non-target) must have shrunk to less than 10 mm in the short axis. The term "complete response" (CR) as used here in relation to non-target lesions refers to the disappearance of all non-target lesions and normalization of tumor marker levels. All lymph nodes must be non-pathogenic in size (<10 mm in the short axis).

[0047] The term "partial response" (PR) used here in relation to target lesions refers to a reduction of at least 30% in the sum of diameters of the target lesions, relative to the baseline sum of diameters.

[0048] As used herein in relation to target lesions, the term “progressive disease” (PD) refers to an increase of at least 20% in the sum of diameters of target lesions, relative to the minimum sum in the study (which includes the baseline sum if it is the minimum in the study). In addition to a relative increase of 20%, the sum must also show an absolute increase of at least 5 mm. The appearance of one or more new lesions is also considered progression. As used herein in relation to non-target lesions, the term “progressive disease” (PD) refers to the appearance of one or more new lesions and / or apparent progression of an existing non-target lesion. Appearant progression is usually not superior to the state of the target lesions. It must represent a change in the overall disease state, rather than a single increase in lesions.

[0049] In relation to the target lesion, the term "stable disease" (SD) used here refers to a state where, based on the minimum diameter sum during the study, there is neither sufficient contraction to qualify as partial reduction (PR) nor sufficient increase to qualify as progressive disease (PD).

[0050] The term "progression-free survival" (PFS) as used herein refers to the period from the start of treatment to disease progression or death, whichever comes first.

[0051] The term "bacterial effector proteins pathogenic to eukaryotic cells" as used herein refers to bacterial effector proteins that are transported to host cells by secretion systems and there exert their pathogenic activity against various host proteins and cellular mechanisms. Many different effector proteins are known, transported by various secretion system types, and exhibit a broad repertoire of biochemical activities that modulate the function of host regulatory molecules. Secretion systems include type 3 (T3SS), type 4 (T4SS), and type 6 (T6SS). Importantly, several effector proteins pathogenic to eukaryotic cells (such as Sigella flexneri IpaC) also belong to the class of bacterial proteins that are part of the secretion system mechanism. If a bacterial effector protein pathogenic to eukaryotic cells is also essential for the function of the secretion system, such a protein is excluded from this definition. T3SS effector proteins that are pathogenic to eukaryotic cells refer to proteins such as Y. enterocolitica YopE, YopH, YopJ, YopM, YopO, YopP, YopT, or Sigella flexneri OspF, IpgD, IpgB1, or Salmonella enterica SopE, SopB, SptP, or Pseudomonas aeruginosa ExoS, ExoT, ExoU, ExoY, or Escherichia coli Tir, Map, EspF, EspG, EspH, EspZ. T4SS effector proteins that are pathogenic to eukaryotic cells include Legionella pneumophila LidA, SidC, SidG, SidH, SdhA, SidJ, SdjA, SdeA, SdeA, SdeC, LepA, LepB, WipA, WipB, YlfA, YlfB, VipA, VipF, VipD, VpdA, VpdB, DrrA, LegL3, LegL5, LegL7, LegLC4, LegLC8, LegC5, LegG2, Ceg10, Ceg23, Ceg29 or Bartonella hensella BepA, BepB, BepC, BepD, BepE, BepF This refers to proteins such as BepG, Agrobacterium tumefaciens VirD2, VirE2, VirE3, VirF, Helicobacter pylori CagA, or Bordetella pertussis pertussis toxin.T6SS effector proteins that are pathogenic to eukaryotic cells refer to proteins such as Vibrio cholerae VgrG proteins (VgrG1, etc.).

[0052] The terms "pathogenic T3SS effector proteins to eukaryotic cells" or "pathogenic bacterial T3SS effector proteins to eukaryotic cells" as used herein refer to proteins spontaneously injected into the cytosol of eukaryotic cells by the T3S apparatus, and proteins spontaneously secreted by the T3S apparatus, which may, for example, form transition pores into the eukaryotic cell membrane, and are pathogenic factors to eukaryotic cells, i.e., proteins that paralyze or reprogram eukaryotic cells for the benefit of the pathogen. Effectors exhibit a broad repertoire of biochemical activities, for example, regulating the function of important host regulatory mechanisms such as phagocytosis and actin cytoskeleton, inflammatory signaling, apoptosis, endocytosis, or secretory pathways. 2,9, but not limited to, AvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrD1, AvrPphB, AvrPphC, AvrPphEPto, AvrPp iBPto, AvrPto, AvrPtoB, AvrRpm1, AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB , GtgA, GtgE, GALA protein family, HopAB2, HopAO1, HopI1, HopM1, HopN1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopU1, HsvB, IcsB, IpaA, IpaH, IpaH7.8, IpaH9.8, IpgB1, IpgB2, IpgD, LcrV, Map, OspC1, Os pE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXo1, PthXo6, PthXo7, SifA, SifB, SipA / SspA, S lrP, SopA, SopB / SigD, SopD, SopE, SopE2, SpiC / SsaB, SptP, SpvB, SpvC, SrfH, SrfJ, Sse, SseB, SseC , SseD, SseF, SseG, SseI / SrfH, SseJ, SseK1, SseK2, SseK3, SseL, SspH1, SspH2, SteA, SteB, SteC, St Including eD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA.

[0053] The Yersinia T3SS effector genes that are pathogenic to eukaryotic cells and can be deleted / mutated from, for example, Y. enterocolitica, are YopE, YopH, YopM, YopO, YopP (also called YopJ), and YopT. 10Each effector gene that is pathogenic to eukaryotic cells can be deleted / mutated from Sigella flexneri (e.g., OspF, IpgD, IpgB1), Salmonella enterica (e.g., SopE, SopB, SptP), Pseudomonas aeruginosa (e.g., ExoS, ExoT, ExoU, ExoY), or Escherichia coli (e.g., Tir, Map, EspF, EspG, EspH, EspZ). The nucleic acid sequences of these genes are available to those skilled in the art, for example, in the Genebank database (yopH, yopO, yopE, yopP, yopM, yopT from NC_002120 GI:10955536; S. flexneri effector protein from AF386526.1 GI:18462515; S. enterica effector from NC_016810.1 GI:378697983 or FQ312003.1 GI:301156631; Pseudomonas aeruginosa effector from AE004091.2 GI:110227054 or CP000438.1 GI:115583796 and Escherichia coli effector protein from NC_011601.1 GI:215485161).

[0054] For the purposes of this invention, genes are indicated in lowercase italics to distinguish them from proteins. When a gene (indicated in lowercase italics) is followed by a bacterial species name (such as E. coli), it refers to a mutation in the corresponding gene in the corresponding bacterial species. For example, YopE refers to the effector protein encoded by the yopE gene. Y. enterocolitica yopE represents Y. enterocolitica having a mutation in the yopE gene.

[0055] Where used herein, the terms "polypeptide," "peptide," "protein," "polypeptide," and "peptide" are interchangeable and refer to a series of amino acid residues linked to one another by peptide bonds between the α-amino and carboxyl groups of adjacent residues. Proteins having an amino acid sequence comprising at least 10 amino acids, more preferably at least 20 amino acids, are preferred.

[0056] According to the present invention, “heterogeneous proteins or fragments thereof” include naturally occurring proteins or fragments thereof, as well as artificially manipulated proteins or fragments thereof. As used herein, the term “heterogeneous proteins or fragments thereof” refers to proteins or fragments thereof other than the T3SS effector protein or its N-terminal fragment that can be fused thereto. In particular, as used herein, heterogeneous proteins or fragments thereof refer to proteins or fragments thereof that do not belong to the proteome of specific recombinant Gram-negative bacterial strains provided and used by the present invention, i.e., proteins or fragments thereof that do not belong to the entire natural protein complement, for example, the proteome of specific strains of the genera Yersinia, Escherichia coli, Salmonella, or Pseudomonas, i.e., proteins or fragments thereof that do not belong to the entire natural protein complement. Typically, heterogeneous proteins or fragments thereof are of animal origin, including human origin. Preferably, heterogeneous proteins or fragments thereof are human proteins or fragments thereof. More preferably, the heterologous protein or fragment thereof is selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, low molecular weight GTPases, GPCR-related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins, or fragments thereof. Particularly preferably, the heterologous protein or fragment thereof is selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, reporter proteins, low molecular weight GTPases, GPCR-related proteins, nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors and viral proteins, or fragments thereof.Even more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, nanobody fusion constructs, and nanobodies. Even more particularly preferred are heterologous proteins or fragments thereof selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, cell cycle regulators, and ankyrin repeat proteins, or fragments thereof. Even more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, ankyrin repeat proteins, cell signaling proteins, nanobody fusion constructs, and nanobodies. Even more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or apoptosis regulation, ankyrin repeat proteins, nanobody fusion constructs, and nanobodies. Furthermore, more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or the regulation of apoptosis, and cell cycle regulators. Furthermore, more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or the regulation of apoptosis, nanobody fusion constructs, and nanobodies. Furthermore, more particularly preferred heterologous proteins or fragments thereof are selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or the regulation of apoptosis, and nanobody fusion constructs.Most preferred are proteins or fragments thereof involved in apoptosis or apoptosis regulation, or proteins or fragments thereof involved in the induction or regulation of the interferon (IFN) response, particularly proteins or fragments thereof involved in the induction or regulation of the interferon (IFN) response, such as heterologous proteins or fragments thereof from animals, preferably humans, involved in apoptosis or apoptosis regulation, or human proteins or fragments thereof involved in the induction or regulation of the interferon (IFN) response. The proteins or fragments thereof involved in the induction or regulation of the interferon (IFN) response are preferably proteins or fragments thereof involved in the induction or regulation of the type I interferon (IFN) response, and more preferably human proteins or fragments thereof involved in the induction or regulation of the type I interferon (IFN) response.

[0057] In some embodiments, the Gram-negative bacterial strain of the present invention comprises two nucleotide sequences encoding identical or two different heterologous proteins or fragments thereof, which are fused in-frame and independently to each other at the 3' ends of a nucleotide sequence encoding a delivery signal from a bacterial effector protein.

[0058] In some embodiments, the Gram-negative bacterial strain of the present invention comprises three nucleotide sequences encoding identical or three different heterogeneous proteins or fragments thereof, which are independently fused in-frame to the 3' ends of a nucleotide sequence encoding a delivery signal from a bacterial effector protein. In some embodiments, the Gram-negative bacterial strain of the present invention comprises four nucleotide sequences encoding identical or four different heterogeneous proteins or fragments thereof, which are independently fused in-frame to the 3' ends of a nucleotide sequence encoding a delivery signal from a bacterial effector protein.

[0059] Heterogeneous proteins expressed by recombinant Gram-negative bacterial strains typically have molecular weights between 1 kDa and 150 kDa, preferably between 1 kDa and 120 kDa, more preferably between 1 kDa and 100 kDa, and most preferably between 10 kDa and 80 kDa. Heterogeneous protein fragments typically contain 10 to 1500 amino acids, preferably 10 to 800 amino acids, more preferably 100 to 800 amino acids, and particularly 100 to 500 amino acids. Heterogeneous protein fragments as defined herein typically possess the same functional properties as the heterogeneous protein from which they originate. The term “heterogeneous protein from which it originates” in relation to a fragment refers to the full-length heterogeneous protein from which the fragment originates. The term “same functional properties as the heterogeneous protein from which it originates” refers to the molecular function (or one of the molecular functions) of the full-length protein from which the fragment originates, which may be, for example, enzyme activity, protein-protein interactions, and / or function as a scaffold protein. Examples of protein fragments involved in the induction or regulation of interferon (IFN) responses include human cGAS and human RIG-I. Full-length human cGAS is a nucleotidyltransferase that catalyzes the formation of cyclic GMP-AMP (cGAMP) from ATP and guanosine triphosphate (GTP). Fragments of human cGAS with "the same functional properties" should be able to perform the same enzymatic activity (cGAMP synthesis from GTP and ATP in the case of cGAS). The same applies to human RIG-I, a cytoplasmic sensor for short double-stranded RNA composed of an RNA helicase domain, a C-terminal domain, and an N-terminal domain (Brisse and Ly, 2019). The helicase domain is involved in the recognition of double-stranded RNA; the C-terminal domain contains a repressor domain; and the N-terminal domain contains two caspase mobilization domains (CARDs) that activate downstream signaling pathways. Therefore, a fragment of human RIG-I possessing the "same functional properties" should be able to perform the same activity as one of the molecular functions of full-length RIG-I, and in the case of the N-terminal CARD domain, it should activate the corresponding downstream signaling pathway.

[0060] In some embodiments, the fragment of a heterologous protein includes a domain of the heterologous protein. Accordingly, in some embodiments, the Gram-negative bacterial strain of the present invention includes a nucleotide sequence encoding a domain of the heterologous protein. Preferably, the Gram-negative bacterial strain of the present invention includes a nucleotide sequence encoding one or two domains of the heterologous protein, more preferably two domains of the heterologous protein.

[0061] In some embodiments, the Gram-negative bacterial strain of the present invention includes a nucleotide sequence encoding a repeat domain of a heterologous protein or two or more domains of different heterologous proteins, which is fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein.

[0062] The term "heterogeneous proteins belonging to the same functional class of proteins" as used herein refers to heterogeneous proteins that have the same function, such as heterogeneous proteins with specific enzymatic activity, heterogeneous proteins that act in the same pathway, such as cell cycle regulation, or heterogeneous proteins that share common specific characteristics, such as belonging to the same class of bacterial effector proteins. Functional classes of proteins include, for example, proteins involved in apoptosis or apoptosis regulation, proteins that act as cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, proteins involved in the induction or regulation of interferon (IFN) responses, reporter proteins, transcription factors, proteases, small GTPases, GPCR-related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors, or viral proteins that act together in biological processes that establish pathogenicity against eukaryotic cells.

[0063] According to the present invention, “heterogeneous protein domains” include domains of naturally occurring proteins, as well as domains of artificially manipulated proteins. As used herein, the term “heterogeneous protein domains” refers to heterogeneous protein domains other than the domain of the T3SS effector protein, or domains other than those containing an N-terminal fragment that can be fused to it to achieve a fusion protein. In particular, as used herein, heterogeneous protein domains refer to the proteome of specific recombinant Gram-negative bacterial strains provided and used by the present invention, i.e., heterogeneous protein domains that do not belong to the whole natural protein complement, for example, the proteome of specific strains of the genera Yersinia, Escherichia coli, Salmonella, or Pseudomonas, i.e., heterogeneous protein domains that do not belong to the whole natural protein complement. Typically, heterogeneous protein domains are of animal origin, including human origin. Preferably, heterogeneous protein domains are human protein domains. More preferably, the heterologous protein domain is a protein domain selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, transcription factors, proteases, low molecular weight GTPases, GPCR-related proteins, nanobody fusion constructs and nanobodies, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins. Particularly preferred, the heterologous protein domain is a protein domain selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators, ankyrin repeat proteins, reporter proteins, low molecular weight GTPases, GPCR-related proteins, nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins.More particularly preferred are heterologous protein domains selected from the group consisting of proteins involved in apoptosis or apoptosis regulation, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators, and ankyrin repeat proteins. Most preferred are protein domains involved in the induction or regulation of interferon (IFN) response, such as animal proteins involved in the induction or regulation of interferon (IFN) response, and more preferably, human heterologous protein domains involved in the induction or regulation of interferon (IFN), particularly human heterologous protein domains involved in the induction or regulation of type 1 interferon (IFN) response.

[0064] The domains of heterologous proteins expressed by recombinant Gram-negative bacterial strains typically have a molecular weight between 1 and 50 kDa, preferably between 1 and 30 kDa, more preferably between 1 and 20 kDa, and most preferably between 1 and 15 kDa.

[0065] According to the present invention, the "proteins involved in the induction or regulation of the IFN response" are not limited to, but include cGAS, STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1 (DDX58), MDA5, LGP2, IPS-1 / MAVS / Cardif / VISA, Trim25, Trim32, Trim56, Riplet, TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, P This includes KR, TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSm14A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, IFNAR1, IFNAR2, TYK2, JAK1, ISGF3, IL10R2, IFNLR1, IFNGR1, IFNGR2, JAK2, STAT4, and cyclic dinucleotide-producing enzymes (cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases), such as WspR, DncV, DisA and DisA-like enzymes, CdaA, CdaS and cGAS or fragments thereof.Preferred proteins involved in inducing or regulating the IFN response include cGAS, STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1(DDX58), MDA5, LGP2, IPS-1 / MAVS / Cardif / VISA, Trim25, Trim32, Trim56, Riplet, TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, TLR3, and TLR 7, selected from the group consisting of TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSm14A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, IFNAR1, IFNAR2, TYK2, JAK1, ISGF3, IL10R2, IFNLR1, IFNGR1, IFNGR2, JAK2, STAT4, cyclic dinucleotide-producing enzymes (cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases), such as WspR, DncV, DisA and DisA-like enzymes, CdaA, CdaS and cGAS, or fragments thereof.

[0066] According to the present invention, the "proteins involved in the induction or regulation of type I IFN response" are not limited to, but include cGAS, STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1, MDA5, LGP2, IPS-1 / MAVS / Cardif / VISA, Trim25, Trim32, Trim56, Riplet, TRAF2, TRAF3, TRAF5, T This includes ANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSm14A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, cyclic dinucleotide-producing enzymes (cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases), such as WspR, DncV, DisA and DisA-like enzymes, CdaA, CdaS and cGAS or fragments thereof.

[0067] Preferred proteins involved in the induction or regulation of type I IFN responses include cGAS, STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1, MDA5, LGP2, IPS-1 / MAVS / Cardif / VISA, Trim25, Trim32, Trim56, Riplet, TRAF2, TRAF3, Selected from the group consisting of cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclase, selected from the group consisting of TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, LSm14A, LRRFIP1, DHX29, DHX15, or fragments thereof, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclase, selected from the group consisting of WspR, DncV, DisA and DisA-like, CdaA, CdaS and cGAS, or fragments thereof.

[0068] More preferred proteins involved in the induction or regulation of type I IFN responses include cGAS (e.g., Uniprot.Q8N884 for human protein), RIG1 (e.g., Uniprot.O95786 for human protein), MDA5 (e.g., Uniprot.Q9BYX4 for human protein), IPS-1 / MAVS (e.g., Uniprot.Q7Z434 for human protein), IRF3 (e.g., Uniprot.Q14653 for human protein), IRF7 (e.g., Uniprot.Q92985 for human protein), IRF9 (e.g., Uniprot.Q00978 for human protein), and cyclic dinucleotide-producing enzymes, such as WspR (e.g., for Pseudomonas aeruginosa). Selected from the group consisting of cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases or fragments thereof, selected from the group consisting of Uniprot.Q9HXT9), DncV (e.g., Uniprot.Q9KVG7 for Vibrio cholerae protein), DisA and DisA-like (e.g., Uniprot.Q812L9 for Bacillus cereus protein), CdaA (e.g., Uniprot.Q8Y5E4 for Listeria monocytogenes protein), CdaS (e.g., Uniprot.O31854 or constitutively active L44F mutation for Bacillus subtilis protein), and cGAS (e.g., Uniprot.Q8N884 for human protein).

[0069] IPS-1 / MAVS / Cardif / VISA refers to a eukaryotic mitochondrial antiviral signaling protein that contains an N-terminal CARD domain and has the Uniprot (www.uniprot.org) identifier "Q7Z434" for the human sequence and "Q8VCF0" for the mouse sequence. The terms "IPS-1 / MAVS," "MAVS / IPS-1," and "MAVS" are used interchangeably here and refer to a eukaryotic mitochondrial antiviral signaling protein that contains an N-terminal CARD domain and has the Uniprot (www.uniprot.org) identifier "Q7Z434" for the human sequence and "Q8VCF0" for the mouse sequence.

[0070] In some embodiments, heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of CARD domain-containing proteins or fragments thereof, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases or fragments thereof. Examples of CARD domain-containing heterologous proteins involved in the induction or regulation of the type I IFN response include RIG1, which typically contains two CARD domains, MDA5, which typically contains two CARD domains, and MAVS, which typically contains one CARD domain.

[0071] The heterologous protein fragments involved in the induction or regulation of the IFN response or type I IFN response typically contain between 25 and 1000 amino acids, preferably between 50 and 600, more preferably between 100 and 500, and even more preferably between 100 and 362 amino acids. In some embodiments, the heterologous protein fragments involved in the induction or regulation of an IFN response or type I IFN response include, typically, a heterologous protein fragment involved in the induction or regulation of an IFN response or type I IFN response, comprising amino acids between 25 and 1000, preferably between 50 and 600, more preferably between 100 and 500, even more preferably between 100 and 362, particularly between 100 and 246; or the heterologous protein fragment involved in the induction or regulation of an IFN response or type I IFN response, comprising amino acid deletions including amino acids between amino acid 1 and amino acid 160 of the N-terminal amino acid sequence, preferably including amino acids 1 to 59 or N-terminal amino acids 1 to 160, and typically comprising amino acids between 25 and 1000, preferably between 50 and 600, more preferably between 100 and 500, even more preferably between 100 and 362.

[0072] Fragments of heterologous proteins containing a CARD domain involved in the induction or regulation of an IFN response or a type I IFN response typically include an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-500, preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-400, more preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-300, more preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid sequence 100-294, and more preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-246.

[0073] In some embodiments, fragments of CARD domain-containing heterologous protein involved in the induction or modulation of the IFN response or type I IFN response are CARD domain-containing heterologous protein, preferably human CARD domain-containing heterologous protein, with amino acid sequences including at least N-terminal amino acid 1 and max amino acid 294, amino acid sequences including at least N-terminal amino acid 1 and max amino acid 246, amino acid sequences including at least N-terminal amino acid 1 and max amino acid 245, amino acid sequences including at least N-terminal amino acid 1 and max amino acid 231, amino acid sequences including at least N-terminal amino acid 1 and max amino acid 229, amino acid sequences including at least N-terminal amino acid 1 and max amino acid 228, and at least N-terminal amino acid 1 and max amino acid 229. The amino acid sequence includes an amino acid sequence containing no acid 218, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 217, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 100, and an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 101, more specifically, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 245, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 228, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 217, and an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 100, most specifically, an amino acid sequence selected from the group consisting of an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 245.

[0074] In some preferred embodiments, the heterologous protein is a fragment of a CARD domain-containing heterologous protein involved in the induction or regulation of an IFN response or a type I IFN response, or comprises a fragment of a CARD domain-containing heterologous protein involved in the induction or regulation of an IFN response or a type I IFN response. Typically, a fragment of a CARD domain-containing heterologous protein involved in the induction or regulation of an IFN response or a type I IFN response comprises at least one CARD domain. In these embodiments, the heterologous protein includes, in particular, an amino acid sequence selected from the group consisting of the amino acid sequences from N-terminal amino acids 1 to 294, N-terminal amino acids 1 to 246, N-terminal amino acids 1 to 245, N-terminal amino acids 1 to 231, N-terminal amino acids 1 to 229, N-terminal amino acids 1 to 228, N-terminal amino acids 1 to 218, N-terminal amino acids 1 to 217, N-terminal amino acids 1 to 100, and N-terminal amino acids 1 to 101 of a heterologous protein involved in the induction or regulation of an IFN response or type I IFN response, including a CARD domain. More specifically, the heterologous protein includes or consists of an amino acid sequence selected from the group consisting of the amino acid sequences from N-terminal amino acids 1 to 245, N-terminal amino acids 1 to 228, N-terminal amino acids 1 to 217, and N-terminal amino acids 1 to 100 of a heterologous protein involved in the induction or regulation of an IFN response or type I IFN response, including a CARD domain.

[0075] In these embodiments, the heterologous protein is more specifically an amino acid sequence selected from the group consisting of the amino acid sequence of N-terminal amino acids 1 to 246, the amino acid sequence of N-terminal amino acids 1 to 245, the amino acid sequence of N-terminal amino acids 1 to 229, the amino acid sequence of N-terminal amino acids 1 to 228, the amino acid sequence of N-terminal amino acids 1 to 218, and the amino acid sequence of N-terminal amino acids 1 to 217 of RIG-1, particularly the amino acid sequence of N-terminal amino acids 1 to 245, the amino acid sequence of N-terminal amino acids 1 to 228, the amino acid sequence of N-terminal amino acids 1 to 217, most specifically the amino acid sequence of N-terminal amino acids 1 to 245, or An amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to amino acid 100 of MAVS and the amino acid sequence from N-terminal amino acid 1 to amino acid 101, or an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to amino acid 294 of MDA5 and the amino acid sequence from N-terminal amino acid 1 to amino acid 231, and more specifically, an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to amino acid 245 of RIG-1, the amino acid sequence from N-terminal amino acid 1 to amino acid 228, and the amino acid sequence from N-terminal amino acid 1 to amino acid, or An amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to amino acid 100 of MAVS, or the amino acid sequence from N-terminal amino acid 1 to amino acid 294 and the amino acid sequence from N-terminal amino acid 1 to amino acid 231 of MDA5. It includes or consists of.

[0076] The most preferred amino acid sequences are those of human RIG-1 from N-terminal amino acid 1 to amino acid 245 and those of mouse RIG-1 from N-terminal amino acid 1 to amino acid 246. The human RIG-1 1-245 fragment and the mouse RIG-1 1-246 fragment correspond to each other with 73% sequence identity (and 85% sequence similarity), and they are functionally equivalent; that is, both fragments exhibit equivalent activity in mouse and human cells.

[0077] In some preferred embodiments, the heterologous protein is a fragment of a cyclic dinucleotide-producing enzyme such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclase. The fragment of a cyclic dinucleotide-producing enzyme such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclase typically includes an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-600, preferably an amino acid sequence from amino acid 50 to any amino acid 100-550, more preferably an amino acid sequence from amino acid 60 to any amino acid 100-530, particularly an amino acid sequence from amino acid 60 to amino acid 530, more specifically an amino acid sequence from amino acid 146 to amino acid 507, or an amino acid sequence from amino acid 161 to amino acid 522, most specifically an amino acid sequence from amino acid 161 to amino acid 522. In some embodiments, the cGAS fragment includes, in particular, an amino acid sequence selected from the group consisting of an amino acid sequence containing at least amino acid 60 and a maximum amino acid 422, an amino acid sequence containing at least amino acid 146 and a maximum amino acid 507, and an amino acid sequence containing at least amino acid 161 and a maximum amino acid sequence 522. In some embodiments, the cGAS fragment includes, more specifically, an amino acid sequence selected from the group consisting of an amino acid sequence from amino acid 60 to amino acid 422, an amino acid sequence from amino acid 146 to amino acid 507, and an amino acid sequence from amino acid 161 to amino acid 522, most preferably an amino acid sequence from amino acid 161 to amino acid 522.

[0078] In a more preferred embodiment, the heterologous protein involved in inducing or regulating the type I IFN response is selected from the group consisting of CARD domain-containing RIG1, MDA5, and MAVS or fragments thereof (where each fragment contains at least one CARD domain), and cGAS and fragments thereof, and in particular, is selected from the group consisting of CARD domain-containing RIG1 and fragments thereof (where each fragment contains at least one CARD domain), CARD domain-containing MAVS and fragments thereof (where each fragment contains at least one CARD domain), and cGAS and fragments thereof. Fragments of these proteins outlined above are particularly preferred. In these more preferred embodiments, CARD domain-containing RIG1, MDA5, and MAVS include a naturally occurring CARD domain and optionally additionally, following the naturally occurring CARD domain, for example, in the case of RIG-1, a fragment containing a naturally occurring helicase domain or fragment thereof, preferably 1 to 500, more preferably 1 to 250, and even more preferably 1 to 150 amino acids (wherein the naturally occurring helicase domain or fragment thereof is non-functional, i.e., does not bind to the CARD domain), or in the case of MAVS, optionally a downstream C-terminal sequence or fragment thereof, preferably 1 to 500, more preferably 1 to 250, and even more preferably 1 to 150 amino acids. In these embodiments, cGAS and its fragments typically include a naturally occurring synthase domain (NTase core and C-terminal domain; 65 As described in Uniprot.Q8N884 for human proteins, the human cGAS contains amino acids 160-522, preferably the cGAS and its fragments contain the naturally occurring synthase domain, but with partial or complete deletion of the N-terminal domain, preferably the complete N-terminal helical extension (N-terminal helical extension; 65 Described in [reference], it has a deletion of amino acids 1-160 of a human cGAS, such as Uniprot.Q8N884 for human proteins. A partial or complete deletion of the N-terminal domain is preferably a deletion of amino acids 1-59.

[0079] In a preferred embodiment, heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of the RIG-I-like receptor (RLR) family (e.g., RIG1 and MDA5) and / or fragments thereof, other CARD domain-containing proteins involved in antiviral signaling and type I IFN induction (e.g., MAVS) and / or fragments thereof, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases selected from the group consisting of WspR, DncV, DisA and DisA-like, CdaA, CdaS and cGAS, and / or fragments thereof, which result in STING stimulation. The term "other CARD domain-containing proteins and fragments thereof involved in antiviral signaling and type I IFN induction" includes MAVS, CRADD / RAIDD, RIPK2 / RIP2, CARD6, NOD1 and NOD2, or fragments thereof. Accordingly, in a more preferred embodiment, heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of the RIG-I-like receptor (RLR) family (e.g., RIG1 and MDA5) or fragments thereof, other CARD domain-containing proteins involved in antiviral signaling and type I IFN induction, selected from the group consisting of MAVS, CRADD / RAIDD, RIPK2 / RIP2, CARD6, NOD1 and NOD2, or fragments thereof, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP cyclases selected from the group consisting of WspR, DncV, DisA and DisA-like, CdaA, CdaS and cGAS, or fragments thereof, which result in STING stimulation.

[0080] In some embodiments, heterologous proteins involved in inducing or regulating the type I IFN response are selected from the group consisting of RIG1, MDA5, LGP2, MAVS, WspR, DncV, DisA and DisA-like proteins, CdaA, CdaS and cGAS or fragments thereof, more preferably selected from the group consisting of RIG1, MAVS, MDA5, WspR, DncV, DisA-like proteins and cGAS or fragments thereof, and most preferably selected from the group consisting of RIG1 or a fragment thereof and cGAS or a fragment thereof.

[0081] In a more preferred embodiment, the protein involved in inducing or regulating the type I IFN response is selected from the group consisting of RIG1, MDA5, MAVS, WspR, DncV, DisA and DisA-like proteins, CdaA, and cGAS or fragments thereof, and more preferably selected from the group consisting of RIG1, MDA5, MAVS, WspR, DncV, DisA-like proteins, CdaA, and cGAS or fragments thereof, and particularly selected from the group consisting of RIG1, MDA5, MAVS, and cGAS or fragments thereof. Fragments of these proteins as described above are particularly preferred.

[0082] In this more preferred embodiment, the RIG1, MDA5, and MAVS fragments typically include an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-500, preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-400, and more preferably an amino acid sequence from N-terminal amino acid 1 to any amino acid 100-300.

[0083] In this more preferred embodiment, the RIG1 fragment includes an amino acid sequence selected from the group consisting of an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 246, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 245, an amino acid sequence containing N-terminal amino acid 1 and max amino acid 229, an amino acid sequence containing N-terminal amino acid 1 and max amino acid 228, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 218, and an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 217, in particular an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 245; the MDA5 fragment includes an amino acid sequence selected from the group consisting of an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 294, an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 231, and the MAVS fragment includes an amino acid sequence selected from the group consisting of an amino acid sequence containing at least N-terminal amino acid 1 and max amino acid 100, and an amino acid sequence containing N-terminal amino acid 1 and max amino acid 101.

[0084] In this more preferred embodiment, the fragment of RIG1 is more specifically an amino acid sequence from N-terminal amino acid 1 to amino acid 246, an amino acid sequence from N-terminal amino acid 1 to amino acid 245, an amino acid sequence from N-terminal amino acid 1 to amino acid 229, an amino acid sequence from N-terminal amino acid 1 to amino acid 228, an amino acid sequence from N-terminal amino acid 1 to amino acid 218, and an amino acid sequence from N-terminal amino acid 1 to amino acid 217, and more specifically an amino acid sequence from N-terminal amino acid 1 to amino acid 245, an amino acid sequence from N-terminal amino acid 1 to amino acid 228, and an N-terminal amino acid sequence. The MDA5 fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences from amino acid 1 to amino acid 217, most specifically from the group consisting of amino acid sequences from N-terminal amino acid 1 to amino acid 245; the MDA5 fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences from N-terminal amino acid 1 to amino acid 294 and amino acid sequences from N-terminal amino acid 1 to amino acid 231; and the MAVS fragment comprises an amino acid sequence selected from the group consisting of amino acid sequences from N-terminal amino acid 1 to amino acid 100 and amino acid sequences from N-terminal amino acid 1 to amino acid 101.

[0085] In this more preferred embodiment, the cGAS fragment typically includes an amino acid sequence of human cGAS from N-terminal amino acid 1 to any amino acid 100-600, preferably an amino acid sequence from amino acid 50 to any amino acid 100-550, more preferably an amino acid sequence from amino acid 60 to any amino acid 100-530, particularly an amino acid sequence from amino acid 60 to amino acid 530, an amino acid sequence from amino acid 146 to amino acid 507, or an amino acid sequence from amino acid 161 to amino acid 530, more specifically an amino acid sequence from amino acid 60 to amino acid 530, or an amino acid sequence from amino acid 161 to amino acid 530.

[0086] In this more preferred embodiment, the cGAS fragment includes, in particular, an amino acid sequence selected from the group consisting of an amino acid sequence comprising at least amino acid 60 and a maximum amino acid 422, an amino acid sequence comprising at least amino acid 146 and a maximum amino acid 507, and an amino acid sequence comprising at least amino acid 161 and a maximum amino acid 522.

[0087] In this more preferred embodiment, the cGAS fragment more specifically includes an amino acid sequence selected from the group consisting of an amino acid sequence from amino acid 60 to amino acid 422, an amino acid sequence from amino acid 146 to amino acid 507, an amino acid sequence from amino acid 161 to amino acid 522, and most specifically an amino acid sequence from amino acid 161 to amino acid 522.

[0088] In a more preferred embodiment, the protein involved in inducing or regulating the type I IFN response is the human RIG1 CARD domain. 1-245 (Sequence ID: 1), Human RIG1 CARD domain 1-228 (Sequence ID: 2), Human RIG1 CARD domain 1-217 (Sequence ID: 3), mouse RIG1 CARD domain 1-246 (Sequence ID: 4), Mouse RIG1 CARD domain 1-229 (Sequence ID: 5), mouse RIG1 CARD domain 1-218 (Sequence ID: 6), Human MAVS CARD Domain 1-100 (Sequence ID: 7), Mouse MAVS CARD domain 1-101 (Sequence ID: 8), N. vectensis cGAS (Sequence ID: 9), Human cGAS 161-522 (Sequence ID: 10), Mouse cGAS 146-507 (Sequence ID: 11), N. bectensis cGAS 60-422 (Sequence ID: 12), Mouse MDA5 1-294 (Sequence ID: 13), Mouse MDA5 1-231 (Sequence ID: 14), Human MDA5 1-294 (Sequence ID: 15), and human MDA5 1-231 Selected from the group consisting of (Sequence ID: 16).

[0089] In a particularly preferred embodiment, the protein involved in inducing or regulating the type I IFN response is the human RIG1 CARD domain. 1-245 (Sequence ID: 1), Human RIG1 CARD domain 1-228 (Sequence ID: 2), Human RIG1 CARD domain 1-217 (Sequence ID: 3), Human MAVS CARD Domain 1-100 (Sequence ID: 7), and human cGAS 161-522 Selected from the group consisting of (Sequence ID: 10).

[0090] In a more particularly preferred embodiment, the protein involved in inducing or regulating the type I IFN response is the human RIG1 CARD domain. 1-245 (Sequence ID: 1), mouse RIG1 CARD domain 1-246 (Sequence ID: 4), Mouse RIG1 CARD domain 1-229 (Sequence ID: 5), mouse RIG1 CARD domain 1-218 (Sequence ID: 6), and human cGAS 161-522 Selected from the group consisting of (Sequence ID: 10), most specifically the human RIG1 CARD domain 1-245 (Sequence ID: 1) and Human cGAS 161-522 Selected from the group consisting of (Sequence ID: 10).

[0091] The RIG-I-like receptor (RLR) family includes proteins selected from the group consisting of RIG1, MDA5, and LGP2. Preferred heterologous proteins involved in the induction or regulation of type I IFN responses are the CARD domain-containing proteins RIG1 and MDA5, particularly the CARD domain-containing protein RIG1. Other preferred CARD domain-containing proteins involved in type I IFN induction include proteins selected from the group consisting of MAVS.

[0092] In some preferred embodiments, heterologous proteins involved in inducing or regulating the type I IFN response are selected from the group of proteins comprising the CARD domain of RIG1, the CARD domain of MDA5, and / or the CARD domain of MAVS, and WspR, DncV, DisA and DisA-like proteins, CdaA, CdaS and cGAS, and fragments thereof, preferably selected from the group of proteins comprising the CARD domain of RIG1, the CARD domain of MDA5, and / or the CARD domain of MAVS, and WspR, DncV, DisA and DisA-like proteins, CdaA and cGAS, or fragments thereof.

[0093] In some preferred embodiments, the heterologous protein involved in inducing or regulating the type I IFN response is selected from the group consisting of the CARD domain of RIG1, the CARD domain of MDA5, the CARD domain of MAVS, WspR, DncV, DisA and DisA-like proteins, CdaA, CdaS and cGAS, and more preferably from the group consisting of the CARD domain of RIG1, WspR, DncV, DisA-like proteins and cGAS.

[0094] In some preferred embodiments, the heterologous protein involved in inducing or regulating the type I IFN response comprises one or more (e.g., two, three, or four) CARD domains, preferably one or more (e.g., two, three, or four) RIG1, MDA5, and / or MAVS, preferably the CARD domains of RIG1 and / or MAVS. In more preferred embodiments, the heterologous protein involved in inducing or regulating the type I IFN response comprises both CARD domains of RIG1, both CARD domains of MDA5, and / or the CARD domains of MAVS and cGAS or a fragment thereof, particularly both CARD domains of RIG1 and cGAS or a fragment thereof, more specifically both CARD domains of RIG1.

[0095] In some embodiments, heterologous proteins involved in the induction or regulation of type I IFN response are selected from the group consisting of type I IFN response-inducing proteins without enzymatic function and type I IFN response-inducing proteins with enzymatic function. Type I IFN response-inducing proteins without enzymatic function as encompassed in the present invention typically include at least one CARD domain, preferably two CARD domains. A CARD domain typically consists of a bundle of 6 to 7 alpha helices, preferably an antiparallel alpha-helix configuration having a hydrophobic core and an outer surface composed of charged residues. Type I IFN response-inducing proteins with enzymatic function as encompassed in the present invention typically include cyclic dinucleotide-producing enzymes (cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases) or their domains that result in STING stimulation, preferably diadenylate cyclase (DAC), diguanylate cyclase (DGC), or GMP-AMP cyclase (GAC) or their domains.

[0096] According to the present invention, "proteins involved in apoptosis or apoptosis regulation" are not limited to, but include Bad, Bcl2, Bak, Bmt, Bax, Puma, Noxa, Bim, Bcl-xL, Apaf1, caspase 9, caspase 3, caspase 6, caspase 7, caspase 10, DFFA, DFFB, ROCK1, APP, CAD, ICAD, CAD, EndoG, AIF, HtrA2, Smac / Diablo, Arts, ATM, ATR, Bok / Mtd, Bmf, Mcl-1(S), IAP family, LC8, PP2B, 1 This includes 4-3-3 proteins, PKA, PKC, PI3K, Erk1 / 2, p90RSK, TRAF2, TRADD, FADD, Daxx, caspase 8, caspase 2, RIP, RAIDD, MKK7, JNK, FLIPs, FKHR, GSK3, CDKs, and their inhibitors, such as the INK4 family (p16 (Ink4a), p15 (Ink4b), p18 (Ink4c), p19 (Ink4d)) and the Cip1 / Waf1 / Kip1-2 family (p21 (Cip1) / Waf1), p27 (Kip1), p57 (Kip2). Preferably, Bad, Bmt, Bcl2, Bak, Bax, Puma, Noxa, Bim, Bcl-xL, caspase 9, caspase 3, caspase 6, caspase 7, Smac / Diablo, Bok / Mtd, Bmf, Mcl-1(S), LC8, PP2B, TRADD, Daxx, caspase 8, caspase 2, RIP, RAIDD, FKHR, CDKs, and their inhibitors, such as the INK4 family. - (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)), most preferably BIM, Bid, cleaved Bid, FADD, caspase 3 (and its subunits), Bax, Bad, Akt, CDKs, and their inhibitors, such as the INK4 family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d)) are used. 11-13In addition, proteins involved in apoptosis or apoptosis regulation include DIVA, Bcl-Xs, Nbk / Bik, Hrk / Dp5, Bid and tBid, Egl-1, Bcl-Gs, cytochrome C, beclin, CED-13, BNIP1, BNIP3, Bcl-B, Bcl-W, Ced-9, A1, NR13, Bfl-1, caspase 1, caspase 2, caspase 4, caspase 5, and caspase 8.

[0097] Proteins involved in apoptosis or its regulation are selected from the group consisting of pro-apoptotic proteins, anti-apoptotic proteins, inhibitors of anti-apoptotic pathways, and inhibitors of pro-survival signaling or pathways. Pro-apoptotic proteins include Bax, Bak, Diva, Bcl-Xs, Nbk / Bik, Hrk / Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1, Smac / Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, cytochrome C, FADD, caspase family, CDKs and their inhibitors, such as the INK4 family (p16 (Ink4a), p15 (Ink4b) The proteins are selected from the group consisting of ), p18(Ink4c), and p19(Ink4d), or include proteins selected from Bax, Bak, Diva, Bcl-Xs, Nbk / Bik, Hrk / Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1, Smac / Diablo, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, cytochrome C, FADD, and the caspase family. Preferred are Bax, Bak, Diva, Bcl-Xs, Nbk / Bik, Hrk / Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Egl-1, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin-1, Egl-1 and CED-13, Smac / Diablo, FADD, caspase family, CDKs and their inhibitors, such as the INK4 family (p16 (Ink4a), p15 (Ink4b), p18 (Ink4c), p19 (Ink4d)). Equally preferred are Bax, Bak, Diva, Bcl-Xs, Nbk / Bik, Hrk / Dp5, Bmf, Noxa, Puma, Bim, Bad, Bid and tBid, Bok, Apaf1, BNIP1, BNIP3, Bcl-Gs, Beclin 1, Egl-1 and CED-13, Smac / Diablo, FADD, and the caspase family.

[0098] Anti-apoptotic proteins include proteins selected from the group consisting of Bcl-2, Bcl-X1, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13, the IAP family, and Bfl-1. Preferred proteins are Bcl-2, Bcl-X1, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13, and Bfl-1.

[0099] Inhibitors of the apoptosis prevention pathway include proteins selected from the group consisting of Bad, Noxa, and Cdc25A. Bad and Noxa are preferred.

[0100] Inhibitors of survival-promoting signaling pathways include proteins selected from the group consisting of PTEN, ROCK, PP2A, PHLPP, JNK, and p38. PTEN, ROCK, PP2A, and PHLPP are preferred.

[0101] In some embodiments, heterologous proteins involved in apoptosis or apoptosis regulation are selected from the group consisting of BH3-only proteins, caspases, and intracellular signaling proteins for death receptor regulation of apoptosis, or fragments thereof. BH3-only proteins or fragments thereof are preferred.

[0102] BH3-only proteins include proteins selected from the group consisting of Bad, BIM, Bid and tBid, Puma, Bik / Nbk, Bod, Hrk / Dp5, BNIP1, BNIP3, Bmf, Noxa, Mcl-1, Bcl-Gs, Beclin 1, Egl-1, and CED-13. Preferred are Bad, BIM, Bid and tBid, and especially tBid.

[0103] Caspases include proteins selected from the group consisting of caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 8, caspase 9, and caspase 10. Preferred are caspase 3, caspase 8, and caspase 9.

[0104] Intracellular signaling proteins involved in the death receptor regulation of apoptosis include proteins selected from the group consisting of FADD, TRADD, ASC, BAP31, GULP1 / CED-6, CIDEA, MFG-E8, CIDEC, RIPK1 / RIP1, CRADD, RIPK3 / RIP3, Crk, SHB, CrkL, DAXX, the 14-3-3 family, FLIP, DFF40 and 45, PEA-15, and SODD. FADD and TRADD are preferred.

[0105] In some embodiments, a Gram-negative bacterial strain contains two heterologous proteins involved in apoptosis or apoptosis regulation, where one protein is an apoptosis-promoting protein and the other is an inhibitor of an apoptosis-preventing pathway, or where one protein is an apoptosis-promoting protein and the other is an inhibitor of a survival-promoting signaling pathway.

[0106] The apoptosis-promoting proteins included in the present invention typically have an alpha-helix structure, preferably a hydrophobic helix surrounded by an amphiphilic helix, and typically contain at least one of the BH1, BH2, BH3, or BH4 domains, preferably at least one BH3 domain. Typically, the apoptosis-promoting proteins included in the present invention do not have enzymatic activity.

[0107] The anti-apoptotic proteins included in the present invention typically have an α-helix structure, preferably a hydrophobic helix surrounded by an amphiphilic helix, and include combinations of different BH1, BH2, BH3, and BH4 domains, preferably combinations of different BH1, BH2, BH3, and BH4 domains containing BH1 and BH2 domains, more preferably BH4-BH3-BH1-BH2, BH1-BH2, BH4-BH1-BH2, or BH3-BH1-BH2 (from N-terminus to C-terminus). In addition, proteins containing at least one BIR domain are also included.

[0108] The inhibitors of the apoptosis prevention pathway included in the present invention typically have an α-helix structure, preferably a hydrophobic helix surrounded by an amphiphilic helix, and usually contain a single BH3 domain.

[0109] The BH1, BH2, BH3, or BH4 domains are typically between about 5 and about 50 amino acids in length. Therefore, in some embodiments, heterologous proteins involved in apoptosis or apoptosis regulation are selected from the group consisting of heterologous proteins involved in apoptosis or apoptosis regulation that are about 5 to about 200, preferably about 5 to about 150, more preferably about 5 to about 100, most preferably about 5 to about 50, and particularly about 5 to about 25 amino acids in length.

[0110] Particularly preferred heterologous proteins are BH3 domains of apoptosis-inducing factor tBID, more specifically, BH3 domains containing sequences selected from the group consisting of SEQ ID NOs: 17-20, preferably SEQ ID NO: 17 or SEQ ID NO: 18.

[0111] Equally preferred are BAX domains containing the BH3 domain of the apoptosis regulator BAX, more specifically, BAX domains containing sequences selected from the group consisting of SEQ ID NOs: 21-24, preferably SEQ ID NO: 21 or SEQ ID NO: 22. Human and mouse sequences are given by SEQ ID NOs, but tBID and BAX BH3 domains of all other species are equally included.

[0112] According to the present invention, "cell cycle regulatory factors" are not limited to, but include cyclins (e.g., cyclin A, cyclin B, cyclin D1, cyclin D2, cyclin D3, cyclin E, cyclin H), cyclin-dependent kinases (CDKs such as CDK1, CDK2, CDK4, CDK6), CDK-activated kinases (such as CDK7), Cdk inhibitors (e.g., the INK4 family including INK4A, INK4B, INK4C, and INK4D), and These include the Cip / Kip family (including p21 (Waf1, Cip1), p27 (Cip2), p57 (Kip2)), CDK substrates (e.g., products of retinoblastoma tumor suppressor genes, NPAT, histone H1, p107, p130), and late-stage accelerating complexes / cyclosomes and cell cycle checkpoint proteins (e.g., Mad1, Mad2, BubR1, BUB1, p53, Mdm2, Cdc25, 14-3-3 protein, Cdc20). Preferred cell cycle regulators are selected from the group consisting of cyclins, cyclin-dependent kinases (CDKs), CDK-activated kinases, Cdk inhibitors, CDK substrates, late-stage accelerating complexes / cyclosomes, and cell cycle checkpoint proteins. More preferred cell cycle regulators are selected from the group consisting of cyclin A, cyclin B, cyclin D1, cyclin D2, cyclin D3, cyclin E, cyclin H, CDK1, CDK2, CDK4, CDK7, INK4A, INK4B, INK4C, INK4D, Waf1 (Cip1 / p21), p27 (Cip2), p57 (Kip2), NPAT, histone H1, p107, p130, Mad1, Mad2, BubR1, BUB1, p53, Mdm2, Cdc25, 14-3-3 protein, and Cdc20.

[0113] According to the present invention, the term "cell signaling protein" is not limited to, I. Cytokine signaling (e.g., cytokines such as IL-2 receptor and interleukin receptors; adapter proteins and kinases such as the JAK family; regulators such as SOCS; transcription factors such as IRF3 or IRF9 or the STAT family; and kinases / downstream targets such as AKT and GRB2), II. Survival factor signaling / death signaling / growth factor or hormone signaling (e.g., kinases such as MAPKins like MEK and ERK, other kinases such as Akt or PI3K or SRC or ATM, and kinase complexes such as mTORComplex1; transcription factors such as Elk1 or c-Myc; GTPases such as Ras; growth factor receptors, which are usually receptor tyrosine kinases such as EGFR; hormone receptors such as estrogen receptors), III. Chemokine signaling (e.g., G protein-coupled receptors such as chemokine receptors / CXCR2, which are usually 7-transmembrane proteins; G protein complexes such as the Gi complex, which consists of Galphai, Gbeta, and Ggamma kinases; phospholipase C, protein kinase C, JAK family, RhoGTPases such as PI3K, CDC42, or RhoA, MAPKs such as JNK, adapter proteins such as mTORComplex1, and signaling factors / regulators; transcription factors such as the STAT family or NFκB; translation initiation factors such as eIF4E) and IV. Extracellular matrix / Wnt / Hedgehog signaling (e.g., WNT ligands such as Wnt1 or Wnt3a, WNT ligand receptors such as Frizzled; LRP coreceptors such as LRP6; kinases such as betacatenin, LGR5, Axin.APC, GSK3 beta, CK1alpha, FAK, and casein; cadherins; ubiquitin ligases such as β-TrCP, inhibitory nuclear complexes composed of TLE, histone deacetylases HDAC; enhancers such as Dvl, TCF, and LEF; coactivators such as p300; Rho GTPases such as Rac1; integrins) It includes.

[0114] Preferred cell signaling proteins are selected from the group consisting of cytokine signaling proteins, survival factor signaling proteins, death signaling proteins, growth factor signaling proteins, hormone signaling proteins, chemokine signaling proteins, and extracellular matrix / Wnt / Hedgehog signaling proteins. More preferred cell signaling proteins are selected from the group consisting of cytokine receptors such as GM-CSF receptor, interferon α / β receptor, interferon γ receptor, CD40 or CD120, or their adapter proteins; interleukin receptors such as IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-21 receptor or IL-18 receptor, or their adapter proteins; kinases such as the JAK family, MAPKinase, PI3K, Akt, and kinase complexes such as mTORComplex; transcription factors such as IRF3 or IRF9, STAT family, or NFκB; and RhoGTPases such as CDC42, RhoA, or Rac1. Even more preferred cell signaling proteins are selected from the group consisting of GM-CSF receptor, interferon α / β receptor, interferon γ receptor, CD40, CD120, or their adapter proteins; IL-2 receptor, IL-7 receptor, IL-12 receptor, IL-21 receptor, IL-18 receptor, or their adapter proteins; JAK family, MAPKinase, PI3K, Akt, mTORComplex, IRF3, IRF9, STAT family, NFkB, CDC42, RhoA, and Rac1.

[0115] According to the present invention, the "reporter protein" includes, but is not limited to, fluorescent proteins (such as GFP), luciferase, or enzyme reporter proteins (such as alkaline phosphatase). A preferred reporter protein is an enzyme reporter protein.

[0116] According to the present invention, "GPCR-related proteins" include, but are not limited to, G protein-coupled receptors (such as CXCR2), G protein complexes (composed of G alpha, G beta, and G gamma), kinases (such as PKA), adapter proteins, and signaling / regulating factors (such as phospholipase, adenylyl cyclase, and phosphodiesterase), kinases (such as protein kinase C PKC or PKA), and transcription factors (such as CREB). Preferred GPCR-related proteins are selected from the group consisting of G protein-coupled receptors, G protein complexes, kinases, adapter proteins, signaling / regulating factors, and transcription factors. More preferred GPCR-related proteins are selected from the group consisting of CXCR2, G protein complexes composed of Gα, Gβ, and Gγ, PKA, phospholipase, adenylyl cyclase, phosphodiesterase, protein kinase C PKC, and CREB.

[0117] According to the present invention, "nanobody fusion proteins" include, but are not limited to, nanobodies fused to a proteolytic domain (such as the N-terminal F-box domain of the Drosophila Slmb protein or a ubiquitin ligase; therefore, a nanobody fused to a cell signaling protein or a portion thereof); nanobodies fused to the same or other nanobodies (dual or multispecific nanobodies); nanobodies fused to a reporter protein; and nanobodies fused to an intracellular localization signal (such as a nuclear localization signal NLS). Preferred nanobody fusion proteins are selected from the group consisting of nanobodies fused to a proteolytic domain, nanobodies fused to a cell signaling protein or a portion thereof, nanobodies fused to the same or other nanobodies, nanobodies fused to a reporter protein, and nanobodies fused to an intracellular localization signal. More preferred nanobody fusion proteins are selected from the group consisting of nanobody fused to the N-terminal F-box domain of the Drosophila Slmb protein, nanobody fused to a ubiquitin ligase, nanobody fused to the same or other nanobody with bi- or multi-specificity, and nanobody fused to an NLS. According to the present invention, "nanobody" includes, but is not limited to, a single antibody composed of a monomeric single-chain variable antibody domain. Camelid VHH fragments are an example of nanobody. Preferred nanobody is a single antibody composed of a monomeric single-chain variable antibody domain.

[0118] Another particularly preferred heterologous protein is a heterologous protein containing a domain of a protein involved in the induction or regulation of type I IFN response, more specifically, a heterologous protein containing a domain of a protein involved in the induction or regulation of type I IFN response, selected from the group consisting of i) the CARD domain of RIG1 containing a sequence selected from the group consisting of SEQ ID NOs: 1 to 6, ii) the CARD domain of MDA5 containing a sequence selected from the group consisting of SEQ ID NOs: 13 to 16, preferably SEQ ID NOs: 15 or 16, and iii) the CARD domain of MAVS containing a sequence selected from the group consisting of SEQ ID NOs: 7 or 8, preferably SEQ ID NOs: 7. Another particularly preferred heterologous protein is N. bectensis cGAS (SEQ ID NO: 9), human cGAS 161-522 (Sequence ID: 10), N. bectensis cGAS 60-422 (Sequence ID: 12) or mouse cGAS 146-507 Full-length cGAS such as (SEQ ID NO: 11). The most particularly preferred heterologous proteins are the CARD domain of human RIG1 (SEQ ID NO: 1-3), especially the CARD domain of human RIG1 (SEQ ID NO: 1), and human cGAS. 161-522 It is a heterogeneous protein containing (SEQ ID NO: 10).

[0119] In some embodiments, the heterologous protein is a prodrug-converting enzyme. In these embodiments, the recombinant Gram-negative bacterial strain expresses, and preferably expresses and secretes, the prodrug-converting enzyme. The prodrug-converting enzyme referred to herein is an enzyme that converts a non-toxic prodrug into a toxic drug, preferably an enzyme selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase, β-galactosidase, carboxylesterase, nitroreductase, carboxypeptidase, and β-glucuronidase, more preferably an enzyme selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase, and β-galactosidase.

[0120] The term "protease cleavage site" as used herein refers to a specific amino acid motif within an amino acid sequence, such as a protein or fusion protein, that is cleaved by a particular protease that recognizes that amino acid motif. For an overview, see: 14 See below. Examples of protease cleavage sites are amino acid motifs that are cleaved by proteases selected from the group consisting of enterokinase (light chain), enteropeptidase, precision protease, human rhinovirus protease (HRV 3C), TEV protease, TVMV protease, factor Xa protease, and thrombin.

[0121] The following amino acid motifs are recognized by their respective proteases: - Asp-Asp-Asp-Asp-Lys: Enterokinase (light chain) / Enteropeptidase (SEQ ID NO: 45) - Leu-Glu-Val-Leu-Phe-Gln / Gly-Pro:Prescission protease / Human rhinovirus protease (HRV 3C) (SEQ ID NO: 46) - Modification motif based on Glu-Asn-Leu-Tyr-Phe-Gln-Ser (SEQ ID NO: 47) and Glu-XX-Tyr-X-Gln-Gly / Ser (where X is any amino acid) recognized by TEV protease (tobacco Hetch virus) (SEQ ID NO: 48) - Glu-Thr-Val-Arg-Phe-Gln-Ser:TVMV protease (SEQ ID NO: 49) - Ile-(Glu or Asp)-Gly-Arg: Factor Xa protease (SEQ ID NO: 50) - Leu-Val-Pro-Arg / Gly-Ser: Thrombin (Sequence ID: 51).

[0122] The protease cleavage site used here is ubiquitin. Therefore, in some preferred embodiments, ubiquitin is used as the protease cleavage site, i.e., the nucleotide sequence can be cleaved at the N-terminal site by a specific ubiquitin processing protease, for example, encoding ubiquitin as a protease cleavage site that can be endogenously cleaved at the N-terminal site by a specific ubiquitin processing protease called a deubiquitinating enzyme in cells to which the fusion protein has been delivered. Ubiquitin is processed at its C-terminus by a group of endogenous ubiquitin-specific C-terminal proteases (deubiquitinating enzymes, DUBs). Cleavage of ubiquitin by DUBs is thought to occur precisely at the C-terminus (after G76) of ubiquitin.

[0123] "Individual," "Subject," or "Patient" is a vertebrate. In a given embodiment, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (e.g., mice and rats). In a preferred embodiment, the subject is a human.

[0124] The term "mutation" is used here as a general term and includes changes in both single and multiple base pairs. Such mutations may include substitutions, frameshift mutations, deletions, insertions, and truncations.

[0125] The term “nuclear localization signal” as used herein refers to an amino acid sequence that marks a protein for localization into the nucleus of a eukaryotic cell, and preferably includes a viral nuclear localization signal such as SV40 large T antigen-derived NLS (PPKKKRKV) (SEQ ID NO: 52).

[0126] AclI, HindIII, SpI, Sluci, T-M sp509I, PciI, AgeI, BspMI, BfuAI, SexAI, MluI, BceAI, HpyCH4 IV, HpyCH4III, BaeI, BsaXI, AflIII, SpeI, BsrI, BmrI, BglII, AfeI, AluI, StuI, ScaI, ClaI, BspDII, PI-SceI, Swa I I、CspCI、MfeI、BssSI、BmgBI、Pm1I、DraIII、AleI、EcoP15I、PvuII、AlwNI、BtsIMutI、TspRI、NdeI、NlaIII、CviatI andMFlI I、FspEI、XcmI、BstXI、PflMI、BccI、NcoI、BseYI、FauI、SmaI、X maI, TspMI, Nt.CviPII, LpnPI, AciI, SacII, BsrBI, MspI, HpaII 、ScrFI、BssKI、StyD4I、BsaJI、BslI、BtgI、NciI、AvrII、MnlI、 BbvCI、Nb.BbvCI、Nt.BbvCI、SbfI、Bpu10I、Bsu36I、EcoNI、Hpy AV, BstNI, PspGI, StyI, BcgI, PvuI, BstUI, EagI, RsrII, BsiEI 、BsiWI、BsmBI、Hpy99I、MspA1I、MspJI、SgrAI、BfaI、BspCNI、Xh oI、EarI、AcuI、PstI、BpmI、DdeI、SfcI、AflII、BpuEI、SmlI、Av aI, BsoBI, MboII, BbsI, XmnI, BsmI, Nb.BsmI, EcoRI, HgaI, Aat II、ZraI、Tth111IPflFI、PshAI、AhdI、DrdI、Eco53kI、SacI、Bs eRI, PleI, Nt.BstNBI, MlyI, HinfI, EcoRV, MboI, Sau3AI, DpnII BfuCI、DpnI、BsaBI、TfiI、BsrDI、Nb.BsrDI、BbvI、BtsI、Nb.BtsI, BstAPI, SfaNI, SphI, NmeAIII, NaeI, NgoMIV, BglI, AsiSI, BtgZI, HinP1I, HhaI, BssHII, NotI, Fnu4HI, Cac8 I, MwoI, NheI, BmtI, SapI, BspQI, Nt.BspQI, BlpI, TseI, ApeKI, Bsp1286I, AlwI, Nt.AlwI, BamHI, FokI, BtsCI, Hae III, PhoI, FseI, SfiI, Nari, KasI, SfoI, PluTI, AscI, EciI, BsmFI, ApaI, PspOMI, Sau96I, NlaIV, KpnI, Acc65I, Bs aI, HphI, BstEII, AvaII, BanI, BaeGI, BsaHI, BanII, RsaI, CviQI, BstZ17I, BciVI, SalI, Nt.BsmAI, BsmAI, BcoDI, A paLI, BsgI, AccI, Hpy166II, Tsp45I, HpaI, PmeI, HincII, BsiHKAI, ApoI, NspI, BsrFI, BstYI, HaeII, CviKI-1, Eco O109I, PpuMI, I-CeuI, SnaBI, I-SceI, BspHI, BspEI, MmeI, TaqαI, NruI, Hpy188I, Hpy188III, XbaI, BclI, HpyCH4V This refers to short DNA sequences containing several restriction sites for cleavage by restriction endonucleases, such as FspI, PI-PspI, MscI, BsrGI, MseI, PacI, PsiI, BstBI, DraI, PspXI, BsaWI, BsaAI, EaeI, preferably XhoI, XbaI, HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, and BstBI. The term "multiple cloning site" as used herein further refers to short DNA sequences used in recombination events, such as in Gateway cloning strategies, or in methods such as Gibson assembly or topo cloning.

[0127] The terms “wild-type strain” or “wild-type Gram-negative strain” as used herein refer to naturally occurring mutant strains, or naturally occurring mutant strains that include gene modifications that enable the use of vectors, such as deletion mutations in restriction endonucleases or antibiotic resistance genes. These strains include chromosomal DNA and, in some cases (e.g., Y. enterocolitica, S. flexneri) unmodified pathogenic plasmids.

[0128] The term "Yersinia wild-type strain" as used herein refers to naturally occurring mutant strains (such as Y. enterocolitica E40) or naturally occurring mutant strains that have genetic modifications enabling the use of vectors, such as deletion mutations in restriction endonucleases or antibiotic resistance genes (such as Y. enterocolitica MRS40 or an ampicillin-sensitive derivative of Y. enterocolitica E40). These strains contain chromosomal DNA and unmodified pathogenic plasmids (called pYVs).

[0129] Y. enterocolitica subspecies *Palearctica* refers to a low-pathogenic strain of Y. enterocolitica, in contrast to the more pathogenic *Enterocolitica* subspecies. 15,16 Y. enterocolitica subspecies *Palearcutica* lacks a highly pathogenic island (HPI) compared to Y. enterocolitica subspecies *Enterocolitica*. This HPI encodes an iron siderofoam called yersinia bactin. 17 The absence of yersinia bactin in the Y. enterocolitica subspecies paleacutica makes this subspecies less pathogenic, and persistent infection in the liver or spleen becomes dependent on the systemic availability of iron, for example. 17 For example, pretreatment with deferoxamine, an iron chelating agent used to treat iron overload in patients, can make iron available to bacteria in an individual. 18 .

[0130] The term "comprise" and its variants such as "comprises" and "comprising" are generally used in the sense of including, that is, "including but not limited to", that is, allowing the presence of one or more features or components.

[0131] The singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise.

[0132] The term "about" refers to a range of values within ±10% of the specified value. For example, the phrase "about 200" includes ±10% of 200, that is, from 180 to 220.

[0133] In one aspect, the present invention provides: i) a first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or a fragment thereof fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is operably linked to a promoter; ii) a second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or a fragment thereof fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is operably linked to a promoter; iii) a third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or a fragment thereof fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is operably linked to a promoter; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. The present invention provides a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules, wherein the first and second polynucleotide molecules are located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules are located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, provided that the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located.

[0134] In some embodiments, recombinant Gram-negative strains are recombinant attenuated pathogenic Gram-negative strains.

[0135] The recombinant Gram-negative bacterial strain of the present invention can be obtained by the following: 1) A step of transforming a Gram-negative bacterial strain with a polynucleotide molecule, preferably a DNA polynucleotide molecule, comprising a nucleotide sequence encoding a heterologous protein and a nucleotide sequence homologous or identical to a nucleotide sequence encoding a delivery signal from a bacterial effector protein, or a nucleotide sequence homologous or identical to a nucleotide sequence encoding a fragment of a delivery signal from a bacterial effector protein, wherein the delivery signal from the bacterial effector protein or a fragment thereof is encoded on the chromosome or endogenous pathogenic plasmid of the Gram-negative bacterial strain. Preferably, the nucleotide sequence homologous or identical to the nucleotide sequence of the delivery signal from the bacterial effector protein or a fragment thereof is located at the 5' end of the nucleotide sequence encoding the heterologous protein. The nucleotide sequence encoding the heterologous protein may be flanked at the 3' end of the delivery signal from the bacterial effector protein by a nucleotide sequence homologous to the nucleotide sequence or fragment thereof of the chromosome or endogenous pathogenic plasmid. This nucleotide sequence adjacent to the 3' end of the homologous protein may be homologous to a nucleotide sequence or fragment thereof located within 10 kbp on the chromosome or endogenous pathogenic plasmid at the 3' end of the delivery signal from the bacterial effector protein. This nucleotide sequence adjacent to the 3' end of the homologous protein may be homologous to a nucleotide sequence and may be located within the same operon on the chromosome or endogenous pathogenic plasmid as the delivery signal from the bacterial effector protein or fragment thereof. Transformation is typically carried out so that a nucleotide sequence encoding a heterologous protein is inserted at the 3' end of the delivery signal from a bacterial effector protein encoded by a chromosome or endogenous pathogenic plasmid onto the chromosome of an endogenous pathogenic plasmid or recombinant pathogenic attenuated Gram-negative strain, preferably an endogenous pathogenic plasmid, and the heterologous protein fused to the delivery signal is expressed and secreted. 2) Following (or in parallel with) step 1), the recombinant strain obtained in 1) may be transformed with a further polynucleotide molecule, preferably a DNA polynucleotide molecule, containing a nucleotide sequence encoding a heterologous protein and a nucleotide sequence that is homologous or identical to a nucleotide sequence encoding a delivery signal from a bacterial effector protein, or homologous or identical to a nucleotide sequence encoding a fragment of a delivery signal from a bacterial effector protein, the delivery signal from the bacterial effector protein or a fragment thereof encoded on the chromosome of the Gram-negative strain or on an endogenous pathogenic plasmid. The nucleotide sequence that may be homologous or identical to the nucleotide sequence or fragment thereof of the delivery signal from the bacterial effector protein may be located at the 5' end of the nucleotide sequence encoding the heterologous protein. The nucleotide sequence encoding the heterologous protein may be flanked at its 3' end by a nucleotide sequence homologous to the nucleotide sequence or fragment thereof on the chromosome or endogenous pathogenic plasmid at the 3' end of the delivery signal from the bacterial effector protein. This nucleotide sequence adjacent to the 3' end of the homologous protein may be homologous to a nucleotide sequence or fragment thereof located within 10 kbp on the chromosome or endogenous pathogenic plasmid at the 3' end of the delivery signal from the bacterial effector protein. This nucleotide sequence adjacent to the 3' end of the homologous protein may be homologous to a nucleotide sequence and may be located within the same operon on the chromosome or endogenous pathogenic plasmid as the delivery signal from the bacterial effector protein or fragment thereof. Transformation is typically carried out so that a nucleotide sequence encoding a heterologous protein is inserted at the 3' end of the delivery signal from a bacterial effector protein encoded by a chromosome or endogenous pathogenic plasmid onto the chromosome of an endogenous pathogenic plasmid or recombinant pathogenic attenuated Gram-negative strain, preferably an endogenous pathogenic plasmid, and the heterologous protein fused to the delivery signal is expressed and secreted. 3) The recombinant strains obtained in 1) and 2) can be further genetically transformed with one or two polynucleotide constructs, such as an expression vector, which include one (in the case of two vectors) or two (in the case of one vector) nucleotide sequences encoding heterologous proteins and nucleotide sequences homologous or identical to nucleotide sequences encoding delivery signals from bacterial effector proteins, or homologous or identical to nucleotide sequences encoding fragments of delivery signals from bacterial effector proteins. When the recombinant strains obtained in 1) and 2) are transformed with a single vector which includes two nucleotide sequences encoding heterologous proteins and nucleotide sequences homologous or identical to nucleotide sequences encoding delivery signals from bacterial effector proteins, or homologous or identical to nucleotide sequences encoding fragments of delivery signals from bacterial effector proteins, in one embodiment these two sequences can be fused to form an operon.

[0136] The order of steps 1-3) can be changed, or the steps can be combined, without altering the ultimately generated recombinant strain.

[0137] If the recombinant pathogenic attenuated Gram-negative strain is a Yersinia strain, the endogenous pathogenic plasmid is pYV (Yersinia pathogenic plasmid). If the recombinant pathogenic attenuated Gram-negative strain is a Salmonella strain, the endogenous site for insertion is either one of the gene clusters called SpiI or SpiII (Salmonella pathogenic islands), where the effector protein is encoded elsewhere, or one of the Salmonella pathogenic plasmids (SVPs) instead.

[0138] In one embodiment, the third and fourth polynucleotide molecules are inserted into and / or positioned in an endogenous pathogenic plasmid, preferably at the native site of a bacterial effector protein in the endogenous pathogenic plasmid, for example, at the native site of a pathogenic factor, preferably at the native site of YopE and / or YopH, or at the native site of another Yop (YopO, YopP, YopM, YopT), preferably at the native sites of YopE and YopH, respectively, if the recombinant Gram-negative strain is a Yersinia strain, or at the native site of an effector protein encoded in or otherwise encoded in SpiI, SpiII, preferably at the native site of an effector protein encoded in SpiI or SpiII, more preferably at the native site of SopE or SteA, if the recombinant Gram-negative strain is a Salmonella strain.

[0139] In a preferred embodiment, heterologous proteins or fragments thereof encoded by the nucleotide sequences of the first, second, third, and fourth polynucleotide molecules are independently cGAS, STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1, MDA5, LGP2, IPS-1 / MAVS / Cardif / VISA, Trim25, Trim32, Trim56, Riplet, TRAF2, Selected from the group consisting of TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSm14A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, cyclic dinucleotide-producing enzymes (cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases), such as WspR, DncV, DisA and DisA-like enzymes, CdaA, CdaS and cGAS, or fragments thereof. Proteins involved in the induction or regulation of type I IFN response; proteins involved in apoptosis or apoptosis regulation, selected from the group consisting of pro-apoptosis proteins, anti-apoptosis proteins, inhibitors of apoptosis prevention pathways, and inhibitors of survival-promoting signaling or pathways; cell cycle regulators, selected from the group consisting of cyclins, cyclin-dependent kinases (CDKs), CDK-activated kinases, Cdk inhibitors, CDK substrates, late-stage promoting complexes / cyclosomes, and cell cycle checkpoint proteins; ankyrin repeat proteins; cell signaling proteins, selected from the group consisting of cytokine signaling proteins, survival factor signaling proteins, death signaling proteins, growth factor signaling proteins, hormone signaling proteins, chemokine signaling proteins, and extracellular matrix / Wnt / Hedgehog signaling proteins; reporter proteins, selected from the group consisting of fluorescent proteins, luciferases, and enzyme reporter proteins; transcription factors; proteases; small GTPases;GPCR-related proteins selected from the group consisting of G protein-coupled receptors, G protein complexes, kinases, adapter proteins, signaling factors / regulators, and transcription factors; nanobody fusion constructs selected from the group consisting of nanobodies fused to proteolytic domains, nanobodies fused to cell signaling proteins or parts thereof, nanobodies fused to the same or other nanobodies, nanobodies fused to reporter proteins, and nanobodies fused to intracellular localization signals; nanobodies; bacterial T3SS effectors; bacterial T4SS effectors and viral proteins; or selected from the group consisting of fragments thereof.

[0140] In one embodiment, the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the first polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the third polynucleotide molecule encode the same heterogeneous protein or fragment thereof.

[0141] In a further embodiment, the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the second polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the fourth polynucleotide molecule encode the same heterogeneous protein or fragment thereof.

[0142] In a preferred embodiment, the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the first polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the third polynucleotide molecule encode the same heterogeneous protein or fragment thereof, and the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the second polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof of the fourth polynucleotide molecule encode the same heterogeneous protein or fragment thereof, and the heterogeneous protein or fragment thereof encoded by the first and third polynucleotide molecules is different from the heterogeneous protein or fragment thereof encoded by the second and fourth polynucleotide molecules.

[0143] In a more preferred embodiment, the heterologous proteins encoded by the nucleotide sequences of the first and third polynucleotide molecules are independently selected from the RIG-I-like receptor (RLR) family, other CARD domain-containing proteins or fragments thereof involved in antiviral signaling and type I IFN induction, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases, selected from the group consisting of WspR, DncV, DisA and DisA-like, CdaA, CdaS, and cGAS or fragments thereof as described above, which result in STING stimulation. Even more preferably, the heterologous proteins encoded by the nucleotide sequences of the first and third polynucleotide molecules are independently selected from the group consisting of RIG1, MDA5, MAVS, WspR, DncV, DisA and DisA-like, CdaA, and cGAS or fragments thereof as described above. In particular, heterologous proteins encoded by the nucleotide sequences of the first and third polynucleotide molecules are cGAS or fragments thereof, such as the fragments described above, and more specifically, the human cGAS or fragments thereof shown in Sequence ID No. 10.

[0144] In a more preferred embodiment, the heterologous proteins encoded by the nucleotide sequences of the second and fourth polynucleotide molecules are independently selected from the group consisting of the RIG-I-like receptor (RLR) family, other CARD domain-containing proteins or fragments thereof involved in antiviral signaling and type I IFN induction, and cyclic dinucleotide-producing enzymes such as cyclic di-AMP, cyclic di-GMP, and cyclic di-GAMP cyclases, selected from the group consisting of WspR, DncV, DisA and DisA-like, CdaA, CdaS, and cGAS, or fragments thereof as described above, which result in STING stimulation. Even more preferably, the heterologous proteins encoded by the nucleotide sequences of the second and fourth polynucleotide molecules are independently selected from the group consisting of RIG1, MDA5, MAVS, WspR, DncV, DisA and DisA-like, CdaA, and cGAS or fragments thereof. In particular, heterologous proteins encoded by the nucleotide sequences of the second and fourth polynucleotide molecules are independently selected from the group consisting of RIG1, MDA5, MAVS, WspR, DncV, DisA and DisA-like molecules, and CdaA or fragments thereof. More specifically, heterologous proteins encoded by the nucleotide sequences of the second and fourth polynucleotide molecules are RIG1 or the aforementioned fragments thereof, more specifically fragments of RIG1 containing a CARD domain, even more specifically fragments of RIG1 containing two CARD domains, preferably human RIG1, most specifically fragments of human RIG1 containing two CARD domains, such as those shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3, preferably as shown in SEQ ID NO: 1.

[0145] In one embodiment, a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a first polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a second polynucleotide molecule, are each ligated to act on the same promoter. The phrase "ligated to act on the same promoter" means, in this context, that one promoter (the same promoter) drives the expression of the heterologous proteins of the first and second polynucleotide molecules. In a preferred embodiment, a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a first polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a second polynucleotide molecule, are each ligated to act on the same YopE promoter.

[0146] In further embodiments, a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a third polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a fourth polynucleotide molecule, are linked to act on two different promoters. In a preferred embodiment, the nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a third polynucleotide molecule, is linked to the YopE promoter, and the nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a fourth polynucleotide molecule, is linked to the YopH promoter.

[0147] In a more preferred embodiment, a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a first polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a second polynucleotide molecule, are linked to the same promoter, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a third polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a fourth polynucleotide molecule, are linked to two different promoters.

[0148] The vector containing the first and second polynucleotide molecules may be a low-copy-number, medium-copy-number, or high-copy-number plasmid. Low-copy-number plasmids typically have 1 to 15 copies per bacterial cell, preferably 1 to 10 copies per bacterial cell. Medium-copy-number plasmids typically have 5 to 200 copies per bacterial cell, preferably 10 to 150 copies per bacterial cell. High-copy-number plasmids typically have 100 to 1,000 copies per bacterial cell, preferably 150 to 700 copies per bacterial cell.

[0149] In a preferred embodiment, the vector containing the first and second polynucleotide molecules is a medium copy number plasmid. In a preferred embodiment, the vector is a medium copy number plasmid having 5 to 200 copies / bacterial cell, i.e., 5 to 200 copies of the plasmid are present in a single bacterial cell, preferably 10 to 150 copies / bacterial cell, i.e., 10 to 150 copies of the plasmid are present in a single bacterial cell.

[0150] In one embodiment, the vector containing the first and second polynucleotide molecules is a plasmid having a size of 1 to 15 kDa, preferably 2 to 10 kDa, and more preferably 3 to 7 kDa, without the insert.

[0151] In one embodiment, the extrachromosomal genetic element is an endogenous pathogenic plasmid, preferably an endogenous pathogenic plasmid that naturally encodes a protein of the type III secretion system. In a preferred embodiment, the extrachromosomal genetic element is an endogenous pathogenic plasmid pYV.

[0152] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain is selected from the group consisting of the genera Yersinia, Escherichia coli, Salmonella, and Pseudomonas. In one embodiment, the recombinant Gram-negative bacterial strain is selected from the group consisting of the genera Yersinia and Salmonella. Preferably, the recombinant Gram-negative bacterial strain is a Yersinia strain, more preferably a Yersinia enterocolitica strain. Most preferably, Yersinia enterocolitica E40 (O:9, biotype 2)19 , or 20 which is an ampicillin-sensitive derivative thereof such as Y. enterocolitica MRS40 (also called Y. enterocolitica subsp. palearctica MRS40) as described in. As described in Yersinia enterocolitica E40 and 20 the derivative Y. enterocolitica MRS40 as described in 15、17、21 is identical to Y. enterocolitica subsp. palearctica E40 and its derivative Y. enterocolitica subsp. palearctica MRS40 described herein. Also preferably, the recombinant Gram-negative strain is a Salmonella strain, more preferably a Salmonella (Salmonella enterica) strain. Most preferred is Salmonella enterica Serovar Typhimurium SL1344 as deposited with the Public health England culture collection (NCTC 13347).

[0153] In some embodiments of the present invention, the recombinant Gram-negative strain does not produce siderophores, for example is deficient in siderophore production, preferably does not produce siderophores, for example is a strain deficient in the production of any siderophore. Such a strain is, for example, 15、17、20、21 Y. enterocolitica subsp. palearctica MRS40 described herein, which does not produce yersiniabactin and is preferred.

[0154] In one embodiment of the present invention, the delivery signal from a bacterial effector protein comprises a bacterial effector protein or an N-terminal fragment thereof, preferably a bacterial effector protein pathogenic to eukaryotic cells or an N-terminal fragment thereof.

[0155] In one embodiment of the present invention, the delivery signal from a bacterial effector protein is a delivery signal from a bacterial effector protein selected from the group consisting of bacterial T3SS effector protein or its N-terminal fragment, bacterial T4SS effector protein or its N-terminal fragment, and bacterial T6SS effector protein or its N-terminal fragment. In one embodiment of the present invention, the delivery signal from a bacterial effector protein is a bacterial T3SS effector protein including bacterial T3SS effector protein or its N-terminal fragment, and the T3SS effector protein or its N-terminal fragment may include a chaperone binding site. The T3SS effector protein or its N-terminal fragment including a chaperone binding site is particularly useful as a delivery signal in the present invention. Preferred T3SS effector proteins or their N-terminal fragments include SopE, SopE2, SptP, YopE, ExoS, SipA, SipB, SipD, SopA, SopB, SopD, IpgB1, IpgD, SipC, SifA, SseJ, Sse, SrfH, YopJ, AvrA, AvrBsT, YopT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG, OspI, IpaH, SspH1, VopF, ExoS, ExoT, HopAB2, XopD, AvrRpt2, HopAO1, HopPtoD2, HopU1, and GALA proteins. The group is selected from the chlorine family, AvrBs2, AvrD1, AvrBS3, YopO, YopP, YopE, YopM, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspC1, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpm1, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD, and AvrXv3.A more preferred T3SS effector protein or its N-terminal fragment is selected from the group consisting of SopE, SptP, YopE, ExoS, SopB, IpgB1, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO, YopP, YopE, YopM, and YopT, and the most preferred T3SS effector protein or its N-terminal fragment among them is selected from the group consisting of IpgB1, SopE, SopB, SptP, OspF, IpgD, YopH, YopO, YopP, YopE, YopM, YopT, and especially YopE or its N-terminal fragment.

[0156] Equally preferred T3SS effector proteins or their N-terminal fragments are SopE, SopE2, SptP, SteA, SipA, SipB, SipD, SopA, SopB, SopD, IpgB1, IpgD, SipC, SifA, SifB, SseJ, Sse, SrfH, YopJ, AvrA, AvrBsT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG, OspI, IpaH, VopF, ExoS, ExoT, HopAB2, AvrRpt2, HopAO1, HopU1, GALA protein family, A The group selected consists of vrBs2, AvrD1, YopO, YopP, YopE, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspC1, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpm1, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD, and AvrXv3. Equally preferred T3SS effector proteins or their N-terminal fragments are selected from the group consisting of SopE, SptP, SteA, SifB, SopB, IpgB1, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO, YopP, YopE, and YopT, and among these, the most equally preferred T3SS effector proteins or their N-terminal fragments are selected from the group consisting of IpgB1, SopE, SopB, SptP, SteA, SifB, OspF, IpgD, YopH, YopO, YopP, YopE, and YopT, particularly SopE, SteA, or YopE or their N-terminal fragment, more specifically SteA or YopE or their N-terminal fragment, and most specifically YopE or its N-terminal fragment.

[0157] In some embodiments, the delivery signals from the bacterial effector protein to the first, second, third, and fourth polynucleotide molecules are the same delivery signal. In preferred embodiments, the delivery signals from the bacterial effector protein to the first, second, third, and fourth polynucleotide molecules are the delivery signals from the bacterial T3SS effector protein, preferably the same delivery signal from the bacterial T3SS effector protein. In more preferred embodiments, the delivery signals from the bacterial effector protein to the first, second, third, and fourth polynucleotide molecules include the YopE effector protein or its N-terminal fragment.

[0158] In some embodiments, the delivery signal from the bacterial effector protein is encoded by a nucleotide sequence comprising the bacterial effector protein or its N-terminal fragment, the N-terminal fragment comprising at least the first 10, preferably at least the first 20, and more preferably at least the first 100 amino acids of the bacterial T3SS effector protein. The term "at least the first 10 amino acids of the bacterial T3SS effector protein" refers to the first 10 NH2-terminal (also called N-terminal) amino acids of the bacterial T3SS effector protein.

[0159] In some embodiments, the delivery signal from the bacterial effector protein is encoded by a nucleotide sequence comprising the bacterial T3SS effector protein or its N-terminal fragment, the bacterial T3SS effector protein or its N-terminal fragment comprising a chaperone-binding site.

[0160] Preferred T3SS effector proteins or their N-terminal fragments containing a chaperone binding site include the following combinations of the chaperone binding site and the T3SS effector protein or its N-terminal fragment: SycE-YopE, InvB-SopE, SicP-SptP, SycT-YopT, SycO-YopO, SYcN / YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF, SycD-YopB, SycD-YopD. More preferred are SycE-YopE, InvB-SopE, SycT-YopT, SycO-YopO, SycN / YscB-YopN, SycH-YopH, SpcS-ExoS, and CesF-EspF. Most preferably, YopE or its N-terminal fragment containing a SycE chaperone binding site, for example, here YopE 1-138 This is expressed as follows: the N-terminal fragment of the YopE effector protein containing the N-terminal 138 amino acids of the YopE effector protein, as shown in Sequence ID: 25, or the SopE effector protein or its N-terminal fragment containing the InvB chaperone binding site, for example, in this case, SopE 1-81 Or SopE 1-105 This is denoted as such and is an N-terminal fragment of the SopE effector protein, containing the N-terminal 81 or 105 amino acids of the SopE effector protein, as shown in Sequence IDs 26 and 27.

[0161] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain is a Yersinia strain, and the delivery signal from the bacterial effector protein includes the YopE effector protein or its N-terminus, preferably the Y. enterocolitica YopE effector protein or its N-terminus. Preferably, the SycE binding site is contained within the N-terminus of the YopE effector protein. In this regard, the N-terminal fragment of the YopE effector protein may contain N-terminal amino acids 12, 16, 18, 52, 53, 80, or 138. 22~24、68 The most preferable option, for example, here is YopE 1-138 This is indicated by sequence number 25, and refers to Forsberg and Wolf-Watz. 25This is the N-terminal fragment of the YopE effector protein, containing the N-terminal 138 amino acids of the YopE effector protein, as described in [reference].

[0162] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain is a Salmonella strain, and the delivery signal from the bacterial effector protein encoded by the nucleotide sequence includes a SopE or SteA effector protein or its N-terminus, preferably a Salmonella SopE or SteA effector protein or its N-terminus. Preferably, the chaperone binding site is contained within the N-terminus of the SopE effector protein. In this regard, the N-terminal fragment of the SopE effector protein may contain 81 or 105 amino acids at the N-terminus. Most preferred are the full-length SteA (SEQ ID NO: 28) and the N-terminal fragment of the SopE effector protein containing 105 amino acids at the N-terminus of the effector protein, as described, for example, SEQ ID NO: 27.

[0163] Those skilled in the art are familiar with methods for identifying polypeptide sequences of effector proteins capable of delivering proteins. For example, one such method is Sory et al. 19 As described by [translate], in short, polypeptide sequences from various parts of the Yop protein can be fused in-frame to a reporter enzyme such as the calmodulin-activated adenylate cyclase domain (or Cya) of Bordetella pertussis cyclolysine. Delivery of the Yop-Cya hybrid protein to the eukaryotic cell cytosol is indicated by the appearance of cyclase activity in infected eukaryotic cells, resulting in cAMP accumulation. By using such an approach, those skilled in the art can determine, as needed, the minimum sequence requirements for protein delivery, i.e., the shortest continuous amino acid sequence. For example, 19 See [reference]. Accordingly, the preferred delivery signal of the present invention consists of at least the minimum amino acid sequence of a T3SS effector protein capable of delivering the protein.

[0164] In one embodiment, the recombinant Gram-negative strain is deficient in the production of at least one bacterial effector protein, more preferably deficient in the production of at least one bacterial effector protein that is pathogenic to eukaryotic cells, and even more preferably deficient in the production of at least one T3SS effector protein that is pathogenic to eukaryotic cells. In several embodiments, the recombinant Gram-negative strain is deficient in the production of at least one, preferably at least two, more preferably at least three, even more preferably at least four, particularly at least five, and more specifically at least six, of the most specific bacterial effector proteins that are pathogenic to eukaryotic cells. In some embodiments, the recombinant Gram-negative strain is deficient in the production of at least one, preferably at least two, more preferably at least three, even more preferably at least four, particularly at least five, and more specifically at least six, of the most specific functional bacterial effector proteins that are pathogenic to eukaryotic cells, so that the resulting recombinant Gram-negative strain produces less bacterial effector protein, or produces bacterial effector protein to a lesser degree, compared to a non-pathogenic attenuated Gram-negative wild-type strain, i.e., compared to a Gram-negative wild-type strain that normally produces bacterial effector proteins, or no longer produces functional bacterial effector proteins that are pathogenic to eukaryotic cells.

[0165] According to the present invention, such mutant Gram-negative bacterial strains, i.e., recombinant Gram-negative bacterial strains lacking the production of at least one bacterial effector protein, for example, at least one bacterial effector protein that is pathogenic to eukaryotic cells, such mutant Yersinia strains, can be produced by introducing at least one mutation into a gene encoding at least one effector. Preferably, the genes encoding such effectors include YopE, YopH, YopO / YpkA, YopM, YopP / YopJ, and YopT, as far as Yersinia strains are concerned. Preferably, the genes encoding such effectors include, as far as Salmonella strains are concerned, AvrA, CigR, GogB, GtgA, GtgE, PipB, SifB, SipA / SspA, SipB, SipC / SspC, SipD / SspD, SlrP, SopB / SigD, SopA, SpiC / SsaB, SseB, SseC, SseD, SseF, SseG, SseI / SrfH, SopD, SopE, SopE2, SspH1, SspH2, PipB2, SifA, SopD2, SseJ, SseK1, SseK2, SseK3, SseL, SteC, SteA, SteB, SteD, SteE, SpvB, SpvC, SpvD, SrfJ, and SptP. Most preferably, all genes encoding the effectors are deleted. Those skilled in the art can induce mutations in these T3SS effector genes using any number of standard techniques. Sambrook et al. generally describe such techniques. 26 See below.

[0166] According to the present invention, a mutation can be introduced in the promoter region of a gene encoding an effector, such that the expression of such an effector gene is eliminated.

[0167] It is also possible to induce mutations in the coding region of the gene encoding the effector that cause the catalytic activity of the encoded effector protein to disappear. The "catalytic activity" of an effector protein usually refers to its anti-target cell function, i.e., toxicity. Such activity is governed by the catalytic motif in the catalytic domain of the effector protein. Approaches for identifying the catalytic domain and / or catalytic motif of an effector protein are well known to those skilled in the art. For example, 27,28 See below.

[0168] Accordingly, one preferred mutation of the present invention is the deletion of the entire catalytic domain. Another preferred mutation is a frameshift mutation in the effector-coding gene, in which the catalytic domain is absent in the protein product expressed from such a "frameshift" gene. The most preferred mutation is one involving the deletion of the entire coding region of the effector protein. Other mutations, such as small deletions or base pair substitutions caused in the catalytic motif of the effector protein, resulting in the disruption of the catalytic activity of a given effector protein, are also intended by the present invention.

[0169] Mutations induced in the genes of functional bacterial effector proteins can be introduced into specific strains by many methods. One such method involves cloning the mutated gene into a “suicide” vector, which can introduce the mutated sequence into the strain via allele exchange. An example of such a “suicide” vector is: 29 It is described there.

[0170] In this way, mutations occurring in multiple genes can be successfully introduced into a Gram-negative bacterial strain to produce multiple mutant strains, such as hexavalent recombinant strains. The order in which these mutant sequences are introduced is not important. In some situations, it may be desirable to mutate only a portion, rather than all, of the effector genes. Therefore, the present invention further envisions multiple mutant Yersinia strains other than hexavalent Yersinia, such as double, triple, quadruple, and quintuple mutant strains. The secretion and delivery systems of these mutant strains must be intact in order to deliver proteins.

[0171] The preferred recombinant Gram-negative bacterial strain of the present invention is a hexavalent mutant Yersinia strain in which all effector-coding genes (yopH, yopO, yopP, yopE, yopM, yopT) are mutated so that the resulting Yersinia strain no longer produces functional effector proteins. Such a hexavalent mutant Yersinia strain has been named ΔyopH,O,P,E,M,T for Y. enterocolitica. As an example, such a hexavalent mutant can be produced from the Y. enterocolitica MRS40 strain to produce the preferred Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T (also known here as Y. enterocolitica subspecies paleacutica MRS40 ΔyopH,O,P,E,M,T or Y. enterocolitica ΔyopH,O,P,E,M,T). Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T, which lacks yersinia bactin production, is described in International Publication No. 02077249 and was deposited with the Belgian Coordinated Collections of Microorganisms (BCCM) on September 24, 2001, in accordance with the Budapest Convention on the International Recognition of Microbial Deposits for Patent Proceedings, and was assigned accession number LMG P-21013.

[0172] Equally preferred are Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T ΔHairpinI-virF (also known as Y. enterocolitica ΔyopH,O,P,E,M,T ΔHairpinI-virF), which contains a deletion (ΔHairpinI-virF) on the endogenous pathogenic plasmid pYV that removes an RNA hairpin structure or part thereof, such as a deletion of Hairpin I upstream of the gene encoding an endogenous AraC-type DNA-binding protein. Equally preferred are Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T Δasd pYV-asd (also known here as Y. enterocolitica ΔyopH,O,P,E,M,T Δasd pYV-asd), which contains an endogenous pathogenic plasmid pYV (pYV-asd) comprising a deletion of the chromosomal gene encoding asd and a nucleotide sequence containing the gene encoding asd that is ligated to a promoter. Particularly preferred is Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T Δasd ΔHairpinI-virF pYV-asd, which contains both of the above-mentioned modifications (also known here as Y. enterocolitica ΔyopH,O,P,E,M,T Δasd ΔHairpinI-virF pYV-asd).Particularly preferred strains are those lacking the production of a certain siderophore, preferably not producing any siderophore at all, for example, Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T ΔHairpinI-virF (also called Y. enterocolitica ΔyopH,O,P,E,M,T ΔHairpinI-virF), Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T Δasd pYV-asd (here also called Y. enterocolitica ΔyopH,O,P,E,M,T Δasd pYV-asd), or Y. enterocolitica MRS40 ΔyopH,O,P,E,M,T Δasd ΔHairpinI-virF, as is the case with all Y. enterocolitica subspecies paleacutica strains. This is pYV-asd (Y. enterocolitica ΔyopH, O, P, E, M, T Δasd ΔHairpinI-virF, also called pYV-asd). Therefore, equally preferred strains are Y. enterocolitica subspecies palearctica ΔyopH,O,P,E,M,T ΔHairpinI-virF (also called Y. enterocolitica subspecies palearctica ΔyopH,O,P,E,M,T ΔHairpinI-virF), Y. enterocolitica subspecies palearctica ΔyopH,O,P,E,M,T Δasd pYV-asd (here also called Y. enterocolitica ΔyopH,O,P,E,M,T Δasd), or Y. enterocolitica subspecies palearctica ΔyopH,O,P,E,M,T Δasd ΔHairpinI-virF pYV-asd (here, Y. enterocolitica ΔyopH,O,P,E,M,T Δasd ΔHairpinI-virF This is also known as pYV-asd.

[0173] The most preferred strains are the hexavalent mutant Enterocolitica strains (Yersinia enterocolitica) named ΔyopH, O, P, E, M, and T.

[0174] Polynucleic acid constructs, such as vectors, that can be used according to the present invention to transform Gram-negative bacterial strains may depend on the Gram-negative bacterial strain used, as is known to those skilled in the art. Polynucleic acid constructs that can be used according to the present invention include nucleotide sequences, such as expression vectors (including synthetic or otherwise modified versions of endogenous pathogenic plasmids), vectors for chromosomal or pathogenic plasmid insertion, and DNA fragments for chromosomal or pathogenic plasmid insertion. For example, useful expression vectors for Yersinia, Escherichia coli, Salmonella, or Pseudomonas strains are, for example, pUC, pBad, pACYC, pUCP20, and pET plasmids. For example, a useful vector for chromosomal or pathogenic plasmid insertion for Yersinia, Escherichia coli, Salmonella, or Pseudomonas strains is, for example, pKNG101. DNA fragments for chromosomal or pathogenic plasmid insertion refer to methods used in, for example, Yersinia, Escherichia coli, Salmonella, or Pseudomonas strains, such as lambda red gene manipulation. A vector or DNA fragment for chromosome or pathogenic plasmid insertion can insert the nucleotide sequence of the present invention such that, for example, a nucleotide sequence encoding a heterologous protein, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, is linked to the endogenous promoter of a recombinant Gram-negative bacterial strain in an actionable manner. Therefore, when a vector or DNA fragment for chromosome or pathogenic plasmid insertion is used, the endogenous promoter may be encoded on endogenous bacterial DNA (chromosome or plasmid DNA), and only the respective nucleotide sequences are provided by the manipulation vector or DNA fragment for chromosome or pathogenic plasmid insertion.Alternatively, when a vector or polynucleic acid construct for chromosomal or pathogenic plasmid insertion, such as a nucleotide sequence for chromosomal or pathogenic plasmid insertion, is used, the delivery signals from the endogenous promoter and bacterial effector protein may be encoded on the endogenous bacterial DNA (chromosome or plasmid DNA), and the polynucleic acid construct, such as a nucleotide sequence encoding a heterologous protein, will be provided by the vector for chromosomal or pathogenic plasmid insertion, or by a polynucleic acid construct such as a nucleotide sequence for chromosomal or pathogenic plasmid insertion. Therefore, the promoter does not necessarily have to be included in the vector used for transforming recombinant Gram-negative bacterial strains; that is, the recombinant Gram-negative bacterial strains of the present invention can be transformed with a promoter-free vector.

[0175] Preferred vectors, for example, preferred expression vectors for the genus Yersinia, are selected from the group consisting of pBad_Si_1, pBad_Si_2, and pT3P-715, pT3P-716, and pT3P-717. pBad_Si2 contains the endogenous promoters of YopE and SycE derived from purified pYV40, and is a SycE-YopE vector. 1-138 The fragment was constructed by cloning it into the KpnI / HindIII site of pBad-MycHisA (Invitrogen). Further modifications included the removal of the NcoI / BglII fragment of pBad-MycHisA by digestion, Klenow fragment processing, and religation. Furthermore, YopE 1-138 The following cleavage site was added to the 3' end: XbaI-XhoI-BstBI-(HindIII). pBad_Si1 is equivalent to pBad_Si2 but encodes EGFP amplified from pEGFP-C1(Clontech) at the NcoI / BglII site under an arabinose-inducible promoter. Equally preferred is the use of a modified form of the endogenous Yersinia pathogenic plasmid pYV encoding a heterologous protein as a fusion to the T3SS signal sequence.

[0176] Preferred vectors, such as preferred expression vectors for Salmonella, are selected from the group consisting of pT3P_267, pT3P_268, and pT3P_269. These vectors contain the corresponding endogenous promoter and full-length SteA sequence (pT3P_267), SopE 1-81 Fragment (pT3P_268) or SopE 1-105 Plasmids pT3P_267, pT3P_268, and pT3P_269, containing the fragment (pT3P_269), were amplified from Salmonella SL1344 genomic DNA and cloned into the NcoI / KpnI region of pBad-MycHisA(Invitrogen).

[0177] pT3P-715 is a complete synthetic plasmid (de novo synthetic vector) with similar properties to pSi_2, but the corresponding AraC coding region is deleted, and the ampicillin resistance gene (plus 70 bp upstream) is replaced with the chlorampenicol resistance gene 200 bp upstream. For clarification, pT3P-715 contains the endogenous promoters of YopE and SycE of pYV40, making it a SycE-YopE 1-138 The fragment contained the following cleavage site added to the 3' end of YopE1-138: XbaI-XhoI-BstBI-HindIII. This is the origin of replication for pBR322 and the transposable gene Tn9. 67 It is characterized by chloramphenicol acetyltransferase (CAT).

[0178] pBad_Si2 and pT3P-715 are medium-copy number plasmids that have the pBR322(pMB1) origin (SEQ ID NO: 29).

[0179] The derivative pT3P-716 is a high-copy-number plasmid based on a point mutation in the pBR322 origin (SEQ ID NO: 29), which yields the ColE1 origin (SEQ ID NO: 30). High-copy-number plasmids for the expression and delivery of heterologous cargo proteins are based on pT3P-716.

[0180] The derivative pT3P-717 is a low-copy-number plasmid based on the pBR322 origin of replication, similar to pT3P-715, but also includes the rop ("primer repressor") gene (SEQ ID NO: 31). Low-copy-number plasmids for heterologous cargo protein expression and delivery are based on pT3P-717.

[0181] The polynucleotide molecule of the present invention may include other sequence elements, such as a 3' stop sequence (including a stop codon and a polyA sequence), or a gene that confers drug resistance or other elements that enable selection of a transformant that has received the polynucleotide molecule.

[0182] The polynucleotide molecules of the present invention can be transformed into recombinant Gram-negative bacterial strains by many known methods. For the purposes of the present invention, methods of transformation for introducing polynucleotide molecules include, but are not limited to, electroporation, calcium phosphate-mediated transformation, conjugation, or combinations thereof. For example, a polynucleotide molecule located on a vector can be transformed into an initial strain by a standard electroporation procedure. Subsequently, such a polynucleotide molecule on the vector can be transferred from the initial strain to a desired strain by conjugation, a method also known as "mobilization." Transformants (i.e., Gram-negative bacterial strains that have incorporated the vector) can be selected, for example, using antibiotics. These techniques are well known in the art. For example, 19 See below.

[0183] According to the present invention, the promoter ligated to the bacterial effector protein of the recombinant Gram-negative strain of the present invention may be the natural promoter of the T3SS effector protein of the respective strain or a compatible strain, or another natural promoter of the respective or compatible strain, or a promoter used in expression vectors useful in, for example, Yersinia, Escherichia coli, Salmonella, or Pseudomonas strains, such as pUC and pBad. Such promoters include the T7 promoter, the Plac promoter, or the arabinose-inducible Ara-bad promoter.

[0184] If the recombinant Gram-negative strain is a Yersinia strain, the promoter may be derived from a Yersinia biluron gene. A "Yersinia biluron gene" refers to a gene on a Yersinia pYV plasmid whose expression is regulated by both temperature and contact with target cells. Such genes include genes encoding elements of the secretion mechanism (Ysc genes), genes encoding translocators (YopB, YopD, and LcrV), genes encoding regulatory elements (YopN, TyeA, and LcrG), genes encoding T3SS effector chaperones (SycD, SycE, SycH, SycN, SycO, and SycT), and genes encoding effectors (YopE, YopH, YopO / YpkA, YopM, YopT, and YopP / YopJ), as well as other pYV-encoded proteins such as VirF and YadA.

[0185] In preferred embodiments of the present invention, the promoter is the natural promoter of the gene encoding the T3SS functional effector. When the recombinant Gram-negative strain is a Yersinia strain, the promoter is selected from one of YopE, YopH, YopO / YpkA, YopM, and YopP / YopJ. More preferably, the promoter is derived from YopE and / or YopH. Most preferably, the YopE and YopH promoters are used.

[0186] When the recombinant Gram-negative strain is a Salmonella strain, the promoter may be derived from an SpiI or SpiII pathogenic island or from an effector protein encoded elsewhere. Such genes include genes encoding elements of the secretion mechanism, genes encoding translocators, genes encoding regulatory elements, genes encoding T3SS effector chaperones, and genes encoding effectors, as well as other proteins encoded by SPI-1 or SPI-2. In preferred embodiments of the present invention, the promoter is the native promoter of the gene encoding the T3SS functional effector. When the recombinant Gram-negative strain is a Salmonella strain, the promoter is selected from any one of the effector proteins. More preferably, the promoter is derived from SopE, InvB, or SteA.

[0187] In some embodiments, the promoter is an artificial inducible promoter, such as an IPTG-inducible promoter, a photo-inducible promoter, or an arabinose-inducible promoter.

[0188] In one embodiment of the present invention, a recombinant Gram-negative bacterial strain contains a nucleotide sequence encoding a protease cleavage site. The protease cleavage site is typically located between the nucleotide sequence encoding the heterologous protein and the nucleotide sequence encoding the delivery signal on a polynucleotide molecule containing a nucleotide sequence encoding the heterologous protein, which is fused in-frame to the 3' end of the nucleotide sequence encoding the delivery signal from the bacterial effector protein. The creation of a functional and generally applicable cleavage site enables cleavage of the delivery signal after translocation. Since the delivery signal can interfere with the correct localization and / or function of the translocated protein within the target cell, introducing a protease cleavage site between the delivery signal and the protein of interest allows for the delivery of a nearly native protein into the eukaryotic cell. Preferably, the protease cleavage site is an amino acid motif cleaved by a protease selected from the group consisting of enterokinase (light chain), enteropeptidase, precision protease, human rhinovirus protease 3C, TEV protease, TVMV protease, factor Xa protease, and thrombin, or by its catalytic domain, more preferably an amino acid motif cleaved by TEV protease. Equally preferred protease cleavage sites are an amino acid motif cleaved by a protease selected from the group consisting of enterokinase (light chain), enteropeptidase, precision protease, human rhinovirus protease 3C, TEV protease, TVMV protease, factor Xa protease, ubiquitin processing proteases called deubiquitinating enzymes, and thrombin, or by its catalytic domain. The most preferred amino acid motif is one that is cleaved by a TEV protease or a ubiquitin processing protease.

[0189] Therefore, in a further embodiment of the present invention, heterologous proteins are cleaved by a protease from a delivery signal derived from a bacterial effector protein. A preferred cleavage method is: a) The protease is translocated into eukaryotic cells by the recombinant Gram-negative bacterial strain described herein, which expresses a fusion protein containing a delivery signal derived from a bacterial effector protein and the protease as a heterologous protein; or b) The protease is constitutively or transiently expressed in eukaryotic cells. It is a method.

[0190] Typically, recombinant Gram-negative bacterial strains used to deliver desired proteins to eukaryotic cells are different from recombinant Gram-negative bacterial strains used to transfer proteases into eukaryotic cells.

[0191] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain includes a further nucleotide sequence encoding a labeling molecule or a labeling molecule receptor site. The labeling molecule or the further nucleotide sequence encoding a labeling molecule receptor site is typically fused to the 5' or 3' end of the nucleotide sequence encoding the heterologous protein. Preferred labeling molecules or labeling molecule receptor sites are selected from the group consisting of highly sensitive green fluorescent protein (EGFP), coumarin, coumarin ligase receptor sites, resorphine, resorphine ligase receptor sites, and tetracysteine ​​motifs used with FlAsH / ReAsH dyes (life technologies). Most preferred are resorphine and resorphine ligase receptor sites or EGFP. The use of a labeling molecule or labeling molecule receptor site results in the binding of the labeling molecule to the heterologous protein of interest, which is then delivered into the eukaryotic cell, enabling, for example, protein tracking by live-cell microscopy.

[0192] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain includes a further nucleotide sequence encoding a peptide tag. The further nucleotide sequence encoding the peptide tag is typically fused to the 5' or 3' end of the nucleotide sequence encoding a heterologous protein. Preferred peptide tags are selected from the group consisting of Myc tags, His tags, Flag tags, HA tags, Strep tags, or V5 tags, or from combinations of two or more tags from this group. Most preferred are the Myc tag, Flag tag, His tag, and combined Myc and His tags. The use of peptide tags allows for tracking of tagged proteins, for example, by immunofluorescence or Western blotting using anti-tag antibodies. Furthermore, the use of peptide tags allows for affinity purification of the desired protein after secretion into the culture supernatant or transfer into eukaryotic cells, in either case using a purification method suitable for the corresponding tag (e.g., metal chelate affinity purification used with His tags or anti-Flag antibody-based purification used with Flag tags).

[0193] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain includes a further nucleotide sequence encoding a nuclear localization signal (NLS). This further nucleotide sequence encoding the nuclear localization signal (NLS) is typically fused to the 5' or 3' end of a nucleotide sequence encoding a heterologous protein, and the further nucleotide sequence encodes the nuclear localization signal (NLS). Preferred NLSs include the SV40 large T antigen NLS and its derivatives. 30 The group consists of , and other viral NLSs, which are selected from the group. The most preferred are SV40 large T antigen NLS and its derivatives.

[0194] In one embodiment of the present invention, the recombinant Gram-negative bacterial strain includes multiple cloning sites. The multiple cloning sites are typically located at the 3' end of nucleotide sequences encoding delivery signals from bacterial effector proteins, and / or at the 5' or 3' end of nucleotide sequences encoding heterologous proteins. The vector may contain one or more multiple cloning sites. Preferred multiple cloning sites are selected from the restriction enzymes consisting of XhoI, XbaI, HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, SacI, SalI, and BstBI. The most preferred are XbaI, XhoI, BstBI, and HindIII.

[0195] The fusion protein expressed by the recombinant Gram-negative bacterial strain of the present invention is also called a "fusion protein" or "hybrid protein," i.e., a fusion protein or hybrid of a delivery signal and a heterologous protein. The fusion protein may also contain, for example, a delivery signal and two or more different heterologous proteins.

[0196] This invention aims to provide the Gram-negative bacterial strains described herein for use as pharmaceuticals.

[0197] Therefore, in a further embodiment, the present invention is for use as a pharmaceutical, i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. This relates to a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules, wherein the first and second polynucleotide molecules are located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules are located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, provided that the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located.

[0198] The present invention also envisions a method for treating a target cancer, for example, a malignant solid tumor, which includes delivering heterologous proteins as described above to cancer cells, for example, to cells of a malignant solid tumor or cells of the tumor microenvironment.

[0199] Therefore, in a further embodiment, the present invention is for use in a method for treating a target cancer, i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. The method relates to a recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, wherein the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located, and comprises administering the recombinant Gram-negative bacterial strain to a subject in an amount sufficient to treat the subject.

[0200] Similarly, the present invention relates to a method for treating a target cancer, comprising administering the recombinant Gram-negative bacterial strain to the target, wherein the recombinant Gram-negative bacterial strain is administered in an amount sufficient to treat the target.

[0201] Similarly, the present invention relates to the use of the above-mentioned recombinant Gram-negative bacterial strain in the manufacture of a pharmaceutical product for treating a target cancer.

[0202] Similarly, the present invention relates to the use of the above-mentioned recombinant Gram-negative bacterial strains for treating a target cancer.

[0203] The protein may be delivered, i.e., migrated to cancer cells, e.g., malignant solid tumor cells, when administered to a recombinant Gram-negative bacterial strain, or later, for example, after the recombinant Gram-negative bacterial strain has reached the site of a cancer cell, e.g., malignant solid tumor, and / or after reaching the site of a cancer cell, e.g., malignant solid tumor, and after replicating as described above, to cancer cells, e.g., malignant solid tumor cells or cells of the tumor microenvironment. The delivery time can be controlled, for example, by the promoter used to express the heterologous protein in the recombinant Gram-negative bacterial strain. In the first case, either a constitutive promoter or, more preferably, an endogenous promoter of the bacterial effector protein may drive the heterologous protein. In the case of delayed protein delivery, an artificially inducible promoter, such as an arabinose-inducible promoter, may drive the heterologous protein. In this case, arabinose (or the inducer of the corresponding inducible promoter) is administered to the target when the bacteria have reached and accumulated at the desired site. The arabinose then induces bacterial expression of the protein to be delivered.

[0204] Therefore, in one embodiment, the method for treating cancer is, i) Culturing the recombinant Gram-negative bacterial strains described herein; ii) administering the recombinant Gram-negative bacterial strain described in i) to the subject (where a fusion protein containing a delivery signal from a bacterial effector protein and a heterologous protein is expressed by the recombinant Gram-negative bacterial strain and translocated into cancer cells or tumor microenvironment cells); and optionally, iii) Cleaving the fusion protein so that the heterologous protein is cleaved from the delivery signal from the bacterial effector protein within the cancer cell. The recombinant Gram-negative bacterial strains, including [specific strain name], are administered in amounts sufficient to treat the subject.

[0205] Cancer cells for delivering heterologous proteins are typically cancer cells derived from cancers selected from the group consisting of sarcomas, leukemias, lymphomas, multiple myelomas, central nervous system cancers, and malignant solid tumors, which include, but are not limited to, abnormal cell masses that may arise from various tissue types such as the liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lips, oral cavity, esophagus, ovaries, prostate, stomach, testes, thyroid, or lungs, and therefore include malignant solid tumors of the liver, colon, colorectal, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lips, oral cavity, esophagus, ovaries, prostate, stomach, testes, thyroid, or lung. Preferably, cancer cells for delivering heterologous proteins are cancer cells of malignant solid tumors.

[0206] Therefore, in one preferred embodiment, the cancer is a malignant solid tumor, and the method is i) Culturing the recombinant Gram-negative bacterial strains described herein; ii) administering the recombinant Gram-negative bacterial strain described in i) to the subject (where a fusion protein containing a delivery signal from a bacterial effector protein and a heterologous protein is expressed by the recombinant Gram-negative bacterial strain and translocated into cells of a malignant solid tumor or cells within the tumor microenvironment); and optionally, iii) Cleaving the fusion protein so that the heterologous protein is cleaved from the delivery signal from bacterial effector proteins within malignant solid tumor cells. The recombinant Gram-negative bacterial strains, including [specific strain name], are administered in amounts sufficient to treat the subject.

[0207] In some embodiments, at least two fusion proteins, each containing a delivery signal from a bacterial effector protein and a heterologous protein, are expressed by a recombinant Gram-negative bacterial strain and translocated to eukaryotic cells, such as cancer cells, by the method of the present invention.

[0208] Recombinant Gram-negative bacterial strains can be cultured to express fusion proteins containing delivery signals from bacterial effector proteins and heterologous proteins, according to methods known in the art (e.g., FDA, Bacteriological Analytical Manual (BAM), Chapter 8: Yersinia enterocolitica). Preferably, recombinant Gram-negative bacterial strains can be cultured in Brain Heart Infusion Broth, for example, at 28°C. For induction of T3SS and, for example, YopE / SycE promoter-dependent gene expression, bacteria can be grown at 37°C.

[0209] In one embodiment, cancer cells, such as cells of a malignant solid tumor, are brought into contact with two recombinant Gram-negative bacterial strains i), where the first recombinant Gram-negative strain expresses a first fusion protein containing a delivery signal from a bacterial effector protein and a first heterologous protein, and the second recombinant Gram-negative strain expresses a second fusion protein containing a delivery signal from a bacterial effector protein and a second heterologous protein, resulting in the translocation of the first and second fusion proteins into the cells of the malignant solid tumor or the tumor microenvironment. This embodiment provides, for example, an effective method for delivering two different hybrid proteins to a single cell, by co-infection of cancer cells, such as cells of a malignant solid tumor, with two bacterial strains.

[0210] Those skilled in the art can use a variety of assays to determine whether the fusion protein has been successfully delivered. For example, the fusion protein may be detected by immunofluorescence using an antibody that recognizes the fused tag (such as a Myc tag). The determination can also be made based on the enzymatic activity of the delivered protein, for example, 19 This can be based on the assay described by [name of assay provider].

[0211] The present invention also provides a pharmaceutical composition comprising the recombinant Gram-negative strain described herein, optionally comprising a suitable pharmaceutically acceptable carrier. Accordingly, the present invention also provides a pharmaceutical composition comprising the recombinant Gram-negative strain described herein for use in a method of treating cancer, for example a malignant solid tumor, in a subject.

[0212] The recombinant Gram-negative bacterium can be mixed with a suitable pharmaceutically acceptable carrier for convenient and effective administration in an amount sufficient to treat a subject as a pharmaceutical composition. A unit dosage form of the recombinant Gram-negative bacterium or the pharmaceutical composition to be administered comprises, for example, about 10 5 to about 10 10 bacteria per ml, preferably about 10 6 to about 10 9 bacteria per ml, more preferably about 10 7 to 10 9 bacteria per ml, most preferably about 10 8 bacteria in an amount per ml.

[0213] As used interchangeably herein, "an amount sufficient to treat a subject" or "effective amount" means, within the scope of sound medical judgment, an amount of the bacteria (with a reasonable benefit / risk ratio) that is high enough to significantly positively modify the condition to be treated, but low enough to avoid serious side effects. The effective amount of the bacteria will vary depending on the particular objective to be achieved, the age and health condition of the subject to be treated, the duration of treatment, the nature of any concomitant therapy, and the particular bacterium employed. Accordingly, the effective amount of the bacteria is the minimum amount that will produce the desired effect. Generally, about 10 5 to about 10 10 bacteria, for example about 10 5 to about 10 10 bacteria / m 2 body surface, preferably about 10 6 to about 10 9 bacteria, for example about 10 6 to about 10 9 bacteria / m 2 body surface, more preferably about 10 7 to about 10 8Bacteria, for example about 10 7 to about 10 8 bacteria / m 2 body surface, most preferably 10 8 bacteria, for example 10 8 bacteria / m 2 an amount per body surface is administered to a subject.

[0214] A single dose of a recombinant Gram-negative strain administered to a subject, for example a human, for treating cancer such as a malignant solid tumor is usually about 10 4 to about 10 10 bacteria, for example about 10 4 bacteria / m 2 body surface to about 10 10 bacteria / m 2 body surface, preferably about 10 5 to about 10 9 bacteria, for example about 10 5 to about 10 9 bacteria / m 2 body surface, more preferably about 10 6 to about 10 8 bacteria, for example, about 10 6 to about 10 8 bacteria / m 2 body surface, even more preferably about 10 7 to about 10 8 bacteria, for example about 10 7 to about 10 8 bacteria / m 2 body surface, most preferably 10 8 bacteria, for example 10 8 bacteria / m 2 is the total recombinant Gram-negative strain per body surface.

[0215] Examples of substances that can function as pharmaceutical carriers include sugars, e.g., lactose, glucose, and sucrose; starches and their derivatives, e.g., corn starch and potato starch; cellulose and its derivatives, e.g., sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils, e.g., peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa oil; polyols, e.g., propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; pyrogen-free water; isotonic saline; cranberry extract and phosphate buffer; skim milk powder; and other non-toxic, suitable substances used in pharmaceutical formulations, such as vitamin C, estrogen, and echinacea. Wetting agents and lubricants, such as sodium lauryl sulfate, as well as colorants, flavoring agents, lubricants, additives, tableting agents, stabilizers, antioxidants, and preservatives may also be present.

[0216] Methods for administering recombinant Gram-negative bacteria to a target may be selected from the group consisting of intravenous, intratumoral, intraperitoneal, and oral administration. Although this invention is not intended to be limited to specific application methods, intravenous or intratumoral administration of bacteria or pharmaceutical compositions is preferred.

[0217] Depending on the route of administration, it may be necessary to coat the active ingredients, including bacteria, with materials to protect them from the action of enzymes, acids, and other natural conditions that may inactivate them. For administration of bacteria other than parenteral administration, they should be coated with materials that prevent inactivation, or administered together with such materials. For example, bacteria may be co-administered with enzyme inhibitors or liposomes. Enzyme inhibitors include pancreatic trypsin inhibitors, diisopropyl fluorophosphate (DFP), and trazilol. Liposomes include water-in-oil-in-water P40 emulsions, as well as conventional, specially designed liposomes for transporting bacteria such as Lactobacillus or their byproducts to internal targets in host organisms.

[0218] A single bacterium may be administered alone or in combination with a second different bacterium. Any number of different bacteria may be used in combination. "In combination with" means together, substantially simultaneously, or sequentially. The composition may also be administered, for example, in the form of tablets, pills, or capsules, such as freeze-dried capsules containing the bacteria or pharmaceutical composition of the present invention, or as a frozen solution of the bacteria or pharmaceutical composition of the present invention containing DMSO or glycerol. Another preferred application form is a formulation of freeze-dried capsules of the bacteria or pharmaceutical composition of the present invention. Yet another preferred application form is a formulation of heat-dried capsules of the bacteria or pharmaceutical composition of the present invention.

[0219] The recombinant Gram-negative bacteria or pharmaceutical composition to be administered may be administered by injection. Suitable forms for injection include monoceptive suspensions and monoceptive powders for the immediate preparation of monoceptive injection suspensions. In all cases, the form must be monoceptive and fluid enough to allow for easy injection. The form must be stable under manufacturing and storage conditions. The carrier may be a solvent or dispersion medium, for example, containing water, sugar, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures thereof, and vegetable oil. Adequate fluidity can be maintained, for example, by using a coating agent such as lecithin, or, in the case of a dispersant, by maintaining the required particle size. In many cases, it is preferable to include an isotonic agent, such as sugar or sodium chloride. Sustained absorption of the injection composition can be achieved by using absorption-delaying agents, such as aluminum monostearate and gelatin, in the composition.

[0220] In some embodiments of the present invention, recombinant Gram-negative bacterial strains are co-administered to subjects with siderophores. These embodiments are preferred. The siderophores that can be co-administered include hydroxamic acid type, catecholic acid type, and mixed ligand type siderophores. Preferred siderophores are deferoxamine (also known as desferrioxamine B, desferoxamine B, DFO-B, DFOA, DFB, or desferal), desferrioxamine E, deferasirox (Exjade, Desilox, Defriget, Decifer), and deferipron (Feriprox), with deferoxamine being more preferred. Deferoxamine is a bacterial siderophor produced by the Actinomycetes pilosus, and is commercially available, for example, from Novartis Pharma Schweiz AG (Switzerland).

[0221] Co-administration with siderophores may occur before, simultaneously with, or after administration of recombinant Gram-negative bacterial strains. Preferably, siderophores are administered before administration of recombinant Gram-negative bacterial strains, more preferably about 24 hours before, preferably about 6 hours before, more preferably 3 hours before, and particularly 1 hour before, administration of recombinant Gram-negative bacterial strains to the subject. In certain embodiments, the subject is pre-treated with desfreoxamine 1 hour before infection with the recombinant Gram-negative bacterial strain to enable bacterial growth. Typically, siderophores are administered in amounts of about 0.5 × 10⁻⁶. -5 From a mole, approximately 1 x 10 -3 Moles, comfortable approximately 1 x 10 -5 From a mole, approximately 5 x 10 -4 Moles, preferably about 1 × 10⁻⁶ -4 From a mole, approximately 4 x 10 -3 It is co-administered in a single dose of molars. Typically, desferrioxamine is co-administered in a single dose of about 20 mg to about 500 mg per subject, preferably about 50 mg to about 200 mg, and more preferably a single dose of 100 mg of desferrioxamine is administered simultaneously.

[0222] The dosing regimens for the recombinant Gram-negative bacterial strains or pharmaceutical compositions described herein vary, as is known to those skilled in the art, depending on the specific goals to be achieved, the age and health status of the subject, the duration of treatment, the nature of the concomitant therapy, and the specific bacteria used. Recombinant Gram-negative bacterial strains are usually administered to subjects in a single-dose dosing regimen every 1 to 20 days, preferably every 1 to 10 days, and more preferably every 1 to 7 days. The duration of administration is usually about 20 to about 60 days, preferably about 30 to 40 days. Alternatively, the duration of administration is usually about 8 to about 32 weeks, preferably about 8 to about 24 weeks, and more preferably about 12 to about 16 weeks.

[0223] The present invention also provides kits for treating cancer, such as malignant solid tumors, preferably in humans. Such kits generally include recombinant Gram-negative bacterial strains or pharmaceutical compositions described herein, and instructions for using the kit. In some embodiments, the kit includes a carrier, package, or container partitioned to house one or more containers, such as vials, tubes, etc., each container containing one of the distinct elements to be used in the method herein. Suitable containers include, for example, bottles, vials, syringes, and test tubes. In other embodiments, the containers are formed from a variety of materials, such as glass or plastic. [Examples]

[0224] Example 1: A) Materials and methods Bacterial strains and growth conditions. The strains used in this study are listed in Figures 27A to E. These include *E. coli* Top10 used for plasmid purification and cloning, *E. coli* Sm10λpir used for conjugation, and *E. coli* BW19610 used for growing pKNG101. 31The cells were conventionally grown on LB agar plates and in LB broth at 37°C. Ampicillin was used at a concentration of 200 μg / ml (Yersinia) or 100 μg / ml (Escherichia coli), and chloramphenicol was used at 10 μg / ml to select expression vectors. Streptomycin was used at a concentration of 100 μg / ml to select suicide vectors. Y. enterocolitica MRS40 (O:9, biotype 2) 20 Non-ampicillin-resistant E40 derivative 19 The strains derived therefrom were conventionally propagated on Brain Heart Infusion (BHI; Difco) at room temperature. All Y. enterocolitica strains were supplemented with nalidixic acid (35 μg / ml), and all Y. enterocolitica asd strains were further supplemented with 100 μg / ml of meso-2,6-diaminopimeric acid (mDAP, Sigma Aldrich).

[0225] Genetic manipulation of Y. enterocolitica. Genetic manipulation of Y. enterocolitica is described. 32,33 In short, using purified pYV40 plasmid or genomic DNA as a template, mutagenes for gene modification or deletion within the pYV plasmid or on the chromosome were constructed by two-fragment overlap PCR, resulting in 200-250 bp flanking sequences on both sides of the deletion or modification of each gene. Alternatively, complete synthetic DNA fragments (de novo synthesis) with 200-250 bp flanking sequences on both sides of the deletion or modification of each gene were used. The resulting fragments were then used in E. coli BW19610 31 pKNG101 29 The plasmids were cloned. The sequence-validated plasmids were transformed into E. coli Sm10λpir, and from there the plasmids were transferred into the corresponding Y. enterocolitica strains. Mutant strains carrying the embedded vector were grown for several generations without selective pressure. Next, sucrose was used to select for clones that had lost the vector. Finally, the mutant strains were identified by colony PCR. Specific mutagens (pT3P-456, pT3P-457, pT3P-697, and pT3P-714) are listed in Table III.

[0226] Plasmid construction. Plasmids pBad_Si2, pBad_Si1, or pT3P-715 (or derivatives pT3P-716 and pT3P-717) were used to clone fusion proteins having the N-terminal 138 amino acids of YopE (SEQ ID NO: 25). pBad_Si2 (Figure 3) contained endogenous promoters for YopE and SycE from purified pYV40, resulting in SycE-YopE. 1-138 The fragments were constructed by cloning them to the KpnI / HindIII site of pBad-MycHisA (Invitrogen). Additional modifications included removal of the NcoI / BglII fragment of pBad-MycHisA by digestion, Krenow fragmentation, and religation. The bidirectional transcription terminator (BBa_B1006; iGEM foundation) was cloned to either the KpnI-cleaved and Krenow-treated site (pBad_Si2) or the BglII-cleaved site (pBad_Si1). Furthermore, YopE 1-138The following cleavage site was added to the 3' end: XbaI-XhoI-BstBI-(HindIII) (Figure 3B). pBad_Si1 is equivalent to pBad_Si2, but encodes EGFP amplified from pEGFP-C1 (Clontech) at the NcoI / BglII site under an arabinose-inducible promoter. pT3P-715 (Figure 4) is a fully synthetic plasmid (de novo synthetic vector) with similar characteristics to pSi_2, but with the corresponding AraC coding region deleted and the ampicillin resistance gene (plus 70 bp upstream) replaced by a chlorampenicol resistance gene with a 200 bp upstream region. To clarify, pT3P-715 contains the SycE-YopE1-138 fragment, which includes the endogenous promoters of YopE and SycE from pYV40, with the following cleavage site added to the 3' end of YopE1-138: XbaI-XhoI-BstBI-HindIII. This is characterized by the pBR322 replication origin and chloramphenicol acetyltransferase (cat) from the transposing gene element Tn9 (Alton NK, Vapnek D (1979) Nucleotide sequence analysis of the chloramphenicol resistance transposon Tn9. Nature 282:864-869).

[0227] The derivative pT3P-716 (Figure 5) is a high-copy-number plasmid based on a point mutation in the pBR322 origin of replication (SEQ ID NO: 29), which gives rise to the ColE1 origin of replication (SEQ ID NO: 30). The high-copy-number plasmid for the expression and delivery of heterologous cargo proteins is based on pT3P-716. The derivative pT3P-717 (Figure 6) is a low-copy-number plasmid based on the pBR322 origin of replication, similar to pT3P-715, but additionally includes the rop ("primer repressor") gene (SEQ ID NO: 31). The low-copy-number plasmid for the expression and delivery of heterologous cargo proteins is based on pT3P-717. Fully synthesized plasmids (de novo synthetic vectors) of pT3P-716 and pT3P-717.

[0228] Heterogeneous protein for delivery - RIG-I. RIG-I (also known as DDX58; Uniprot O95786 in human protein) is a cytoplasmic sensor for short double-stranded RNA and a major pattern recognition receptor of the innate immune system. RIG-I consists of an RNA helicase domain, a C-terminal domain, and an N-terminal domain (Brisse and Ly, 2019). The helicase domain is crucial for double-stranded RNA recognition; the C-terminal domain contains a repressor domain; and the N-terminal domain contains two caspase-recruiting domains (CARDs) that activate downstream signaling pathways. In its resting state, RIG-I is found with the C-terminal repressor domain covering the RNA-binding and helicase domains. When agonist viral RNA binds to the helicase domain, the protein unfolds, and the N-terminal CARD domain becomes accessible for interaction with downstream partners such as mitochondrial antiviral signaling proteins (MAVS) and tank-binding kinase 1 (TBK1). Following this, nuclear translocation of activating IFN regulator 3 (IRF3) and IRF7 occurs, resulting in the transcription of interferon-stimulated response element (ISRE) regulatory coding sequences such as IFN-α and -β. The heterologous protein for delivery is selected to consist solely of the N-terminal CARD domain of RIG-I (without the rest of the protein; e.g., human RIG-I). 1-245 Or RIG-I 1-229 Or RIG-I 1-218 Since all of them contain both N-terminal CARD domains of RIG-I, they are called RIG-I CARD2 fragments, RIG-1 CARD2, or RIG-1 CARD domains (these are used interchangeably here), resulting in RNA-independent constitutive activation of the RIG-I pathway. The RIG-I CARD domains delivered by bacteria are accessible and result in MAVS and TBK1 activation. This is followed by nuclear translocation of activated IRF3 and IRF7, resulting in transcription of ISRE regulatory coding sequences such as IFNa and β.

[0229] Similarly, the CARD domain(s) of MAVS or MDA5 were selected to function agonist-independently upon bacterial delivery.

[0230] Heterogeneous protein-cGAS for delivery. Cyclic GMP-AMP synthase (cGAS; Uniprot Q8N884 in human protein) is a cytoplasmic sensor for DNA. cGAS is a nucleotidyltransferase that catalyzes the formation of cyclic GMP-AMP (cGAMP) from ATP and guanosine triphosphate (GTP), and is part of the cGAS-STING DNA sensing pathway. It has two major dsDNA binding sites on either side of its catalytic pocket and is activated by binding to cytosolic DNA. After binding to DNA, cGAS acts as a secondary messenger that catalyzes cGAMP synthesis, which then binds to and activates the transmembrane protein 173 (TMEM173) / STING located in the endoplasmic reticulum. STING then activates the protein kinases IκB kinase (IKK) and TBK1, which in turn activate the transcription factors NF-κB and IRF3, inducing interferons and other cytokines. Secondary messenger cGAMPs also pass through to other cells in several ways, thereby transmitting danger signals from cytosolic DNA to surrounding cells. (N-terminus cleaved cGAS (human cGAS)) 161-522 These (etc.) lack an N-terminal DNA-binding domain but retain enzymatic activity. When these cleaved cGAS are delivered into eukaryotic cells, their enzymatic activity generates intracellular cGAMP, leading to activation of the STING pathway. As seen in the RIG-I pathway, activation of the STING pathway ultimately results in the production of type I IFN.

[0231] Human (and other eukaryotic) genes were de novo synthesized and codon usage adapted to Y. enterocolitica (Figure 27A-E), and YopE 1-138The plasmids pBad_Si2, pT3P-715 (medium copy number), pT3P-716 (high copy number), or pT3P-717 (low copy number) were cloned as fusion products (see Table II below). The ligated plasmids were cloned into E. coli Top10. The sequenced plasmids were electroporated into the desired Y. enterocolitica strains using the settings for standard E. coli electroporation.

[0232] JPEG0007914122000001.jpg185150

[0233] JPEG0007914122000002.jpg255165JPEG0007914122000003.jpg34170

[0234] JPEG0007914122000004.jpg242170

[0235] Yop secretion was observed. Yop regulon induction was performed by shifting the culture to 37°C in BHI-Ox (secretion-tolerant conditions). 34 Glucose (4 mg / ml) was added as a carbon source. The whole cells and supernatant fraction were separated by centrifugation at 20800g for 10 minutes at 4°C. The cell pellet was used as the whole cell fraction. Proteins in the supernatant were precipitated with 10% (w / v) trichloroacetic acid at 4°C for 1 hour. After centrifugation (20800g for 15 minutes) and removal of the supernatant, the resulting pellet was washed overnight with ice-cold acetone. The sample was centrifuged again, the supernatant was discarded, the pellet was air-dried, and resuspended in 1× SDS loading dye.

[0236] The secreted proteins were analyzed by SDS-PAGE; in all cases, 3 × 10⁶ dens per lane. 8 Proteins secreted by individual bacteria were loaded. Detection of specific secreted proteins by immunoblotting was performed using a 12.5% ​​SDS-PAGE gel. For detection of proteins within all cells, unless otherwise specified, 2 × 10⁶ lamina per lane were used. 8Individual bacteria were loaded, and proteins were isolated on a 12.5% ​​SDS-PAGE gel and then detected by immunoblotting.

[0237] Immunoblotting is used to detect rat monoclonal antibody against YopE (MIPA193-13A9; 1:1000, 35 The procedure was performed using [a specific method]. Antiserum was pre-absorbed twice overnight against Y. enterocolitica ΔHOPEMTasd to reduce background staining. Detection was performed with a secondary antibody against the rat antibody, conjugated with horseradish peroxidase (1:5000; Southern biotech), before development on ECL chemiluminescent substrate (LumiGlo, KPM).

[0238] Cell culture and infection. B16F1, LN-229, and RAW cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% FCS and 2 mM L-glutamine (cDMEM). THP-1, A20, Jurkat, and 4T1 cells were cultured in RPMI1640 supplemented with 10% FCS and 2 mM L-glutamine. For A20 cells, 1 mM sodium pyruvate, 2.5 / L D-glucose, 10 mM HEPES, and 0.05 mM mercaptoethanol were further added. Y. enterocolitica was grown overnight at room temperature in BHI containing additives and incubated in fresh BHI at 0.2 OD. 600 The solution was diluted to the specified concentration and grown at room temperature for 2 hours. Afterward, the solution was transferred to a 37°C water bath shaker and cultured for a further 30 minutes to 1 hour. Finally, the bacteria were collected by centrifugation (6000 rcf, 30 seconds) and washed once with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. Cells seeded in 96-well or 6-well plates were infected at the indicated MOI in DMEM / RPMI supplemented with 10 mM HEPES and 2 mM L-glutamine. THP-1 cells were differentiated by adding PMA (phorbol myristate acetate) at a final concentration of 20–50 ng / ml for 3–48 hours prior to bacterial infection. After bacterial addition, the plates were centrifuged at 500 g for 1 minute and left at 37°C for the indicated time.

[0239] Direct type I interferon activation assay. Mouse B16F1 melanoma cells, mouse RAW264.7 wild-type macrophages, or human THP-1 monocytes / macrophages that stably express secreted embryonic alkaline phosphatase (SEAP) under the control of the I-ISG54 promoter, which consists of an IFN-inducible ISG54 promoter enhanced by multimerized ISRE, were purchased from InvivoGen (B16-Blue ISG, RAW-Blue ISG, THP1-Blue ISG). Growth conditions and type I IFN assays were adapted from the protocols provided by InvivoGen. In short, 12,500 B16-Blue ISG cells and 30,000 or 100,000 THP-1 cells were seeded in 150 μl of test medium per well in a flat-bottomed 96-well plate (NUNC or Corning) (RPMI + 2 mM L-glutamine + 10% FCS for B16-Blue ISG and PMA-differentiated THP1-Blue ISG cells; DMEM + 2 mM L-glutamine + 10% FCS for RAW-Blue). On the same day or the following day, 15 μl of the desired infection multiplicity (MOI, diluted with test medium) was added per well, followed by short-term centrifugation (500 g, 60 seconds, room temperature) to infect the cells with the target strains. After a 2-hour incubation (37°C and 5% CO2), bacteria were killed by adding test media containing penicillin (100 U / ml) and streptomycin (100 μg / ml). Incubation was continued for 20-24 hours. SEAP and luciferase were detected using QUANTI-Blue. TM and QUANTI-Luc TM The protocol (InvivoGen) was followed. For SEAP detection: 20 μl of cell supernatant was mixed with 180 μl of detection reagent (QUANTI-Blue). TMThe cells were incubated with InvivoGen. The plates were incubated at 37°C, and SEAP activity was measured by reading the OD at 650 nm using a microplate reader (Molecular Devices). As a positive control, mouse IFNγ (stock: 1,000,000 U / ml) diluted to the respective concentrations in test medium was used. For luciferase detection: 50 μl of detection reagent (QUANTI-Luc) was added to 20 μl of cell supernatant. TM InvivoGen was added to an opaque plate (ThermoScientific). Luminescence was immediately measured using a plate reader (BioTek). For activation of LN-229, Jurkat, or A20 cells, 20,000 to 30,000 cells were seeded in 100 μl of test medium per well in a flat-bottomed 96-well plate (NUNC or Corning) (DMEM + 2 mM L-glutamine + 10% FCS or RPMI + 2 mM L-glutamine + 10% FCS + 1 mM sodium pyruvate + 2.5 / L D-glucose + 10 mM HEPES for LNN-29, and an additional 0.05 mM mercaptoethanol for A20 cells). On the same day or the following day, the cells were infected with the target strain by adding 15 μl per well at the desired infection multiplicity (MOI, diluted with test medium). After 4 hours of incubation (37°C and 5% CO2), collect the supernatant and process according to the manufacturer's instructions. TM -Xpress Human IFN-β ELISA or LumiKine TM - The presence of IFNβ was analyzed using Xpress mouse IFN-β ELISA (Invivogen).

[0240] In vivo distribution or efficacy in 4T1, EMT-6, and B16F10 tumor allograft mouse models All animal experiments were approved and conducted in accordance with local guidelines (Tierschutz-Verordnung, Basel-Stadt) and Swiss animal protection laws (Tierschutz-Gesetz) (License 1908; Kantonales Veterinaramt Basel-Stadt). Six-week-old BALB / c (4T1 or EMT-6 model) or C57BL / 6 (B16F10 model) mice were ordered from Janvier Labs. After at least one week of acclimatization, the mice were anesthetized with isoflurane and given 100 μl of 4T1, EMT-6, or B16F10 cells (1 × 10⁶). 5 ~1 × 10 6 The cells were subcutaneously injected into the flank of the mice. During the experiment, the mice's behavior and physical appearance were scored, and their surface temperature and body weight were measured.

[0241] Once the tumor had developed, mice were administered 8 mg / ml of desferal solution (10 ml / kg) by intravenous injection. On the same day or the following day, the mice were administered the corresponding Y. enterocolitica strain (1 × 10⁶ in the 4T1 experiment) by intravenous injection into the tail vein or, in the case of intratumoral (IT) administration, by direct injection into the tumor. 7 Bacteria; in experiment B16F10, 1 × 10 6 (In the EMT-6 and B16F10 experiments, the bacteria used to infect the cells was 7.5 × 10 7 (Bacteria). Inoculum administered to mice was validated by dilution plating. As a control, mice were injected with PBS without endotoxin. In multi-dose experiments, mice were administered 8 mg / ml desferral solution (10 ml / kg) by IP injection 24 hours before the last bacterial treatment. Following tumor progression, tumor length and width were measured with digital calipers. Tumor volume was calculated as 0.5 × length × width 2 It was decided as follows: 1500mm 3A tumor volume exceeding a certain threshold was defined as the humane endpoint. Mice were sacrificed by CO2 inhalation on each post-infection day. Tumors were excised and weighed. Tumors were homogenized. The total CFU of each sample was determined by spotting serial dilutions on LB agar plates containing nalidixic acid (35 μg / ml). To assess the presence of the pYV plasmid, colonies were replicated on LB agar plates containing nalidixic acid (35 μg / ml) and arsenite (400 μM), and the percentage was calculated. To assess the presence of the (medium copy number) vector, colonies were replicated on LB agar plates containing nalidixic acid (35 μg / ml) and chloramphenicol (10 μg / ml), and the percentage was calculated. To assess the presence of both the pYV plasmid and the (medium copy number) vector, colonies were replicated on LB agar plates containing nalidixic acid (35 μg / ml), arsenite (400 μM), and chloramphenicol (10 μg / ml), and the percentages were calculated.

[0242] B) Result Protein delivery system based on type 3 secretion of YopE fusion protein We selected the N-terminal 138 amino acids of YopE (SEQ ID NO: 25) and fused them to the delivered protein. This is because it has been shown to yield the best results for the transfer of other heterologous T3S substrates. 68 Since these N-terminal 138 amino acids of YopE contain CBS, we further decided to co-express SycE. SycE-YopE cloned from purified Y. enterocolitica pYV40 pathogenic plasmid. 1-138 The fragment contains the endogenous promoters of YopE and its chaperone SycE. Thus, SycE and any YopE 1-138 Fusion proteins are induced by a rapid temperature shift from room temperature growth to 37°C. The culture time at 37°C affects the amount of fusion protein present in the bacteria. Multiple cloning sites (MCS) are used in YopE 1-138 The 3' end was appended, followed by the optional addition of Myc and 6xHis tags and a stop codon (SEQ ID NO: 44).

[0243] Background strains were carefully selected. First, to limit the migration of endogenous effectors, we used a Y. enterocolitica strain (named ΔHOPEMT) lacking all known effectors, Yop H, O, P, E, M, and T. 37 .

[0244] Attenuation of pathogenicity due to deficiency / mutation of bacterial effector proteins that have pathogenic activity against eukaryotic cells. In the case of Y. enterocolitica, pathogenicity is reduced by the deficiency of six endogenous effector proteins called "Yersinia extraocular proteins" (Yops), specifically YopH, O, P, E, M, and T (MRS40 pIML421 [yopHΔ1-352, yopOΔ65-558, yopP23, yopE21, yopM23, yopT135]). 37 These Yops are encoded on a plasmid approximately 70 kbp in size, the "Yersinia pathogenic plasmid" (pYV), which encodes the complete type III secretion system (T3SS) and other pathogenic players (Figure 1). YopH, O, P, E, M, and T are the six effector proteins, which are delivered to host cells by the bacterial type III secretion system to modulate and weaken the immune system. Each Yop has a specific biochemical activity in host cells. YopT cleaves the C-terminal cysteine ​​of Rho GTPase, removing the isoprenyl group that anchors the GTPase to the membrane. This inactivation of Rho due to mislocalization allows it to evade phagocytosis by immune cells such as macrophages and neutrophils. 49 In the same pathway, YopE acts as a GTPase-activating protein (GAP) against Rho GTPases, inactivating them. This reduces phagocytosis and inhibits IL-1 beta release by immune cells. 49 Furthermore, YopO acts as a guanidine nucleotide dissociation inhibitor (GDI), inactivating Rho GTPase. YopO also possesses a serine / threonine kinase domain that acts on the actin cytoskeleton in ways that are not yet fully understood.49 YopH is a tyrosine phosphatase that acts on adhesion plaque proteins such as adhesion plaque kinase (Fak) and paxilin, and strongly inhibits phagocytosis by macrophages and neutrophils. 49 YopP, also known as YopJ in Mycobacterium pseudotuberculosis or Bacillus plague, has been found to inactivate the MAPK / NFκB pathway in immune cells, preventing the release of TNFα and IL-8 from immune cells stimulated by the presence of bacteria. Furthermore, YopP has been found to induce apoptosis in immune cells, which may be related to the effect of the MAPK pathway on protecting cells from apoptosis in its activated state. 49 The role of YopM is not yet fully understood, but it has been found to be associated with ribosomal S6 kinase 1 (RSK1) and protein kinase C-like 2 (PRK2). YopM appears to stimulate phosphorylation of RSK1, potentially influencing downstream targets such as cell cycle progression. 49 Deficiting one or more of these Yops dramatically affects the bacterial defense mechanisms against the immune system. 50 Mutations in each yop molecule were confirmed by PCR in their respective regions and by in vitro secretion assays. Analysis of in vitro secretion using SDS-PAGE and Coomassie blue staining confirmed the absence of full-length YopH, O, M, and YopE molecules.

[0245] Furthermore, Y. enterocolitica strains lacking asd (aspartic acid semialdehyde dehydrogenase) were selectively constructed. The mutation in asd leads to a complete loss of growth capacity without the addition of meso-diamino-pimelic acid. This allows for the creation of antibiotic-free plasmid maintenance systems based on the presence of asd on each plasmid. Other trophication-dependent mutants may be used in a similar manner.

[0246] Fusion to the N-terminal secretory signal of bacteria is linked to YopE expressed in bacteria. 1-138The YopE-fused GCN4 leucine zipper (yeast)-WspR GGDEF (Pseudomonas aeruginosa) protein was successfully delivered without interfering with the folding and function of this tripartite protein in eukaryotic cells. This is remarkable because it means that the YopE-fused GCN4 leucine zipper-WspR GGDEF can still dimerize, leading to an active GGDEF domain.

[0247] Delivery of cGAS / STING and RIG-1-like receptor pathway trigger proteins via bacterial T3SS for induction and optimized gene encoding of type I IFN response. In the following section, we used various type I IFN reporter cell lines (as well as natural non-reporter cell lines): • B16F1 mouse melanoma: In the B16F1 reporter cell line, cGAS delivery contributes minimally to IFN induction compared to RIG-1 signaling (Figure 18). Therefore, the B16F1 reporter cell line can be used to evaluate primarily RIG1-dependent signaling and its disruption by additional encoding of cGAS or other STING-activating proteins. • In mouse RAW264.7 macrophages: RAW reporter cell lines, cGAS delivery and RIG signaling activation contribute equally to overall activation (Figure 18). Therefore, RAW reporter cell lines can be used to evaluate the combination of RIG1 and cGAS-dependent signaling and their interference or enhancement by multiple encoding. • Human THP-1 monocytes / macrophages: In the THP-1 reporter cell line, cGAS delivery primarily contributes to IFN induction in THP-1 cells compared to RIG-1 signaling (Figure 18). Therefore, the THP-1 reporter cell line can be used to evaluate primarily cGAS-dependent signaling and its interference by further encoding of RIG-1 or other RLR-activating proteins.

[0248] Fusion protein YopE 1-138 - Human RIG-1 CARD2 (RIG-I 1-245The delivery of cGAS was evaluated for type I IFN induction in a melanoma reporter cell line. The mouse B16F1 melanocyte reporter cell for type I IFN stimulation is based on the activity of secreted alkaline phosphatase under the control of the I-ISG54 promoter, which consists of an IFN-inducible ISG54 promoter enhanced by the multimer ISRE. In this cell line, cGAS delivery contributes minimally to IFN induction in B16F1 cells compared to RIG signaling. Therefore, this reporter cell line can be used to evaluate primarily RIG-dependent signaling and its interference by further encoding of cGAS or other STING-activating proteins. Reporter cells were subjected to YopE 1-138 - Human RIG-1 CARD2 (RIG-I 1-245 ), and YopE 1-138 - Human cGAS 161-522 The bacteria were infected with strains expressing and transferring various amounts (MOI) of YopE. 1-138 -Human RIG-1 CARD2 demonstrated dose-dependent induction of a type I IFN response in this melanoma reporter cell line (Figure 8), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) failed to induce such a response (Figure 8). YopE 1-138 -The activity of human RIG-1 CARD2 was found to be slightly higher when encoded by a medium copy number vector (such as pBad_Si2 or pT3P-715) compared to when encoded by pYV (Figure 8). (cGAS) 161-522 (Delivery of IFN contributes minimally to IFN induction in B16F1 cells) YopE in pYV 1-138 - Human cGAS 161-522 The additional coding is YopE coded in pYV. 1-138 -It was found that it does not impair the activity of human RIG-1 CARD2.

[0249] Therefore, YopE 1-138-The activity of human RIG-1 CARD2 was found to be surprisingly high when encoded by pYV compared to when encoded by the vector, even though a significantly reduced activity should have been expected due to the very low (single) copy number compared to the (medium copy number) vector. Furthermore, YopE on pYV 1-138 - Human cGAS 161-522 The additional coding is YopE coded in pYV. 1-138 -It was found that it did not impair the activity of human RIG-1 CARD2, which is equally surprising, as the presence of the second cargo protein would surely lead to a decrease in the delivery of the first cargo protein.

[0250] Fusion protein YopE 1-138 - Human cGAS 161-522 The delivery of type I IFN was evaluated in RAW macrophage reporter cell lines for type I IFN induction. Reporter cells were selected from YopE 1-138 - Human cGAS 161-522 The bacteria were infected with strains expressing and transferring various amounts (MOI) of YopE. 1-138 - Human cGAS 161-522 This melanoma reporter cell line was shown to induce a dose-dependent type I IFN response (Figure 9), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) could not induce such a response (Figure 9). 1-138 - Human cGAS 161-522 The activity was found to be more strongly increased when encoded by a medium copy number vector (such as pBad_Si2 or pT3P-715) compared to when encoded by pYV (Figure 9).

[0251] Fusion protein YopE 1-138 -Mouse RIG-1 CARD2 (RIG-I 1-246 The delivery of ) was evaluated for type I IFN induction using a melanoma reporter cell line. Mouse B16F1 melanocyte reporter cells were used in YopE 1-138 -Mice were infected with various strains expressing and translocating RIG-1 CARD2 (MOI). YopE 1-138- Mouse RIG-1 CARD2 was shown to dose-dependently induce a type I IFN response in this melanoma reporter cell line (Figure 10), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) could not induce such a response (Figure 10). YopE 1-138 -The activity of mouse RIG-1 CARD2 was found to be slightly higher when encoded by a medium copy number vector (such as pBad_Si2 or pT3P-715) compared to when encoded by pYV (Figure 10). YopE 1-138 -Dual encoding of mouse RIG-1 CARD2 in pYV and medium copy number vectors surprisingly showed a higher signal at a lower MOI than YopE encoded only in medium copy number vectors. 1-138 -It was found to be comparable to the mouse RIG-1 CARD2 (Figure 10).

[0252] YopE 1-138 - Human cGAS 161-522 A fusion protein YopE combined with 1-138 - Human RIG-1 CARD2 (RIG-I 1-245 The delivery of cGAS was evaluated for type I IFN induction in a melanoma reporter cell line. In the mouse B16F1 melanocyte reporter cell line, cGAS 161-522 Delivery contributes minimally to IFN induction in B16F1 cells compared to RIG signaling. Therefore, this reporter cell line is used to primarily analyze RIG-dependent signaling and cGAS 161-522 Alternatively, interference can be evaluated by further encoding of other STING activating proteins. Reporter cells are YopE 1-138 - Human RIG-1 CARD2, and YopE 1-138 - Human cGAS 161-522 The bacteria were infected with strains expressing and transferring various amounts (MOI) of YopE. 1-138 - Human cGAS 161-522 YopE combined with 1-138-Human RIG-1 CARD2 demonstrated dose-dependent induction of a type I IFN response in this melanoma reporter cell line (Figure 11), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) failed to induce such a response (Figure 11). YopE 1-138 - Human RIG-1 CARD2 and YopE 1-138 - Human cGAS 161-522 If both are coded into a medium copy number vector (such as pBad_Si2 or pT3P-715) and pYV, then YopE in the medium copy number vector (such as pBad_Si2 or pT3P-715) and pYV 1-138 - Encoding of human RIG-1 and additionally YopE in pYV 1-138 - Human cGAS 161-522 Compared to encoding only, the activity was found to be identical (Figure 11). (cGAS on the vector) 161-522 (Delivery of YopE contributes minimally to IFN induction in B16F1 cells) 1-138 - Human cGAS 161-522 The additional coding is therefore done by encoding YopE into pYV and vectors. 1-138 -It was found that it does not impair the activity of human RIG-1 CARD2 (Figure 11).

[0253] YopE 1-138 - Human cGAS 161-522 A fusion protein YopE combined with 1-138 - Human RIG-1 CARD2 (RIG-I 1-245 The delivery of IFN was evaluated for type I IFN induction in B16F1 melanoma, RAW macrophages, and human THP-1 reporter cell lines. The B16F1 reporter cell line can be used primarily to evaluate RIG1-dependent signaling, the THP-1 cell line can be used primarily to evaluate cGAS-dependent signaling, and the RAW cell line can be used to evaluate the potential for co-activation of cGAS and RIG-1. Reporter cells were subjected to YopE 1-138 - Human RIG-1 CARD2, and YopE 1-138 - Human cGAS 161-522 The bacteria were infected with strains expressing and transferring various amounts (MOI) of YopE.1-138 - Human cGAS 161-522 YopE combined with 1-138 Human RIG-1 CARD2 demonstrated dose-dependent induction of a type I IFN response in all reporter cell lines (Figure 12), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) was unable to induce such a response (Figure 12).

[0254] In B16F1 cells, which reflect RIG-1-dependent signaling, YopE 1-138 -The strongest signal was observed when human RIG-1 CARD2 was encoded in a medium copy number vector, but not when YopE encoded in pYV. 1-138 -The difference from human RIG-1 CARD2 is small. cGAS on the vector 161-522 The additional coding does not appear to interfere with the delivery and activation of RIG-1 CARD2 (Figure 12).

[0255] In THP-1 cells that reflect cGAS-dependent signaling, YopE 1-138 - Human cGAS 161-522 The strongest signal was observed when it was encoded in a medium copy number vector, and YopE encoded in pYV. 1-138 - Human cGAS 161-522 This is significantly different. Additional coding of RIG-1 CARD2 on the vector is done using cGAS. 161-522 It does not appear to interfere with the delivery and activity of (Figure 12).

[0256] In RAW cells that reflect cGAS and RIG-1-dependent signaling, cGAS 161-522 The signal was found to be equivalent whether RIG-1 CARD2 was encoded by pYV alone or additionally by a (medium copy number) vector (Figure 12). In this cell line, the peak level of activation was reached at a very low MOI, which may explain the equal signal in all test constructs.

[0257] In summary, the endogenous pYV plasmid and, additionally, the human cGAS encoded in a medium copy number vector.161-522 Furthermore, RIG-I CARD2 delivery yielded the best results considering the results from all cell lines tested, and therefore appears to exhibit highly cell type-independent delivery and activity.

[0258] In vivo distribution studies in mouse models of breast cancer: Different bacterial strains were colonized by intravenous administration to mice carrying syngeneic subcutaneous 4T1 mammary tumors, and colonization was evaluated. To validate Y. enterocolitica subspecies Palearcutica MRS40 ΔyopH, O, P, E, M, T and derivatives encoding heterologous type I IFN-inducible proteins (on pYV, on the vector, or on both pYV and the vector), mouse allogeneic tumor studies were conducted using an established 4T1 model of breast cancer (ATCC number CRL-2539). When the subcutaneous tumors reached a predetermined size (approximately 100-200 mm3), the mice were subjected to 1 × 10⁶ transplantation. 7 cfu Y. enterocolitica subspecies paleactica MRS40 ΔyopH, O, P, E, M, T, a control strain that does not deliver cargo, or YopE on an endogenous pYV plasmid. 1-138 - Human cGAS 161-522 and YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 ) encoding, or YopE on both the endogenous pYV plasmid and the medium copy number vector 1-138 - Human cGAS 161-522 and YopE 1-138 - Encoding human RIG-I CARD2, or YopE on both endogenous pYV plasmids and high copy number vectors. 1-138 - Human cGAS 161-522 and human YopE 1-138 - Encoding human RIG-I CARD2, or YopE on both endogenous pYV plasmids and low copy number vectors. 1-138 - Human cGAS 161-522 and YopE 1-138-The cells encoded human RIG-I CARD2 were used for intravenous infection. To enable bacterial growth, mice were pre-treated with desferoxamine 24 hours prior to infection. Mice infected with the attenuated Y. enterocolitica subspecies palearchica MRS40 ΔyopH, O, P, E, M, T strains did not show significant weight loss and were scored normally in terms of physical appearance and behavior even at 6 days post-infection. Bacterial load was determined as colony-forming units CFU per gram of tumor (CFU / g) at 6 days post-infection (Figure 13). In these mice infected with Y. enterocolitica subspecies palearchica MRS40 ΔyopH, O, P, E, M, T, live bacteria were found within malignant solid tumors at 6 days post-infection (Figure 13). Similarly, for all derivative strains derived from Y. enterocolitica ΔyopH,O,P,E,M,T, regardless of whether they encode heterologous type I IFN proteins in pYV, vector, or both pYV and vector, bacteria were found to be viable within malignant solid tumors at 6 days post-infection at levels similar to Y. enterocolitica ΔyopH,O,P,E,M,T. Therefore, YopE 1-138 - Human cGAS 161-522 and YopE 1-138 - It was found that the delivery of RIG-I CARD2 and the copy number of the vector did not alter the bacterial load in solid tumors in a 4T1 breast cancer model (Figure 13).

[0259] YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 The expression and secretion of ) are controlled by YopE 1-138 -The relationship with the copy number of the vector encoding human RIG-I CARD2 was evaluated. For this purpose, in vitro secretion experiments were performed. Expression in bacteria or secretion into the supernatant was evaluated in Y. enterocolitica ΔyopHOPEMT, a control strain that does not deliver cargo, or on low, medium, or high copy number vectors of YopE. 1-138 - We evaluated the gene encoding human RIG-I CARD2. Expression was found to be highest in high copy number plasmids (Figure 14I), which is YopE 1-138-This follows predictions based on the increasing copy number of the vector encoding human RIG-I CARD2. Surprisingly, secretion assays found that secretion was highest with medium and low copy number plasmids, but with high copy number plasmid-based encoding, YopE 1-138 -Secretion of human RIG-I CARD2 was significantly reduced (Figure 14II). Therefore, high copy number plasmids are associated with YopE 1-138 - It appears to increase the expression of human RIG-I CARD2 but decrease its secretory capacity (Figure 14).

[0260] Furthermore, in cell-based assays using the B16F1 reporter cell line, the potential for activation was identified by YopE encoded in pYV. 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 In addition, YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 We evaluated it in relation to the copy number of vectors that only encode (Figure 15). Surprisingly, YopE encoded in pYV 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 In addition to encoding to pYV, YopE is encoded in vectors. 1-138 -Regarding human RIG-I CARD2, type I IFN induction is YopE 1-138 -The weakest result was obtained when encoding human RIG-I CARD2 to a low copy number vector (Figure 15). This is in contrast to secretion analysis (Figure 14), but can be explained by the relationship between encoding to the vector and pYV (as shown in Figure 10) and encoding based only on the vector (as shown in Figure 8). YopE 1-138 - The highest type I IFN induction was observed when human RIG-I CARD2 was encoded from a medium copy number plasmid (Figure 15).

[0261] YopE 1-138 - Human cGAS 161-522 A fusion protein YopE combined with 1-138- Human RIG-1 CARD2 (RIG-I 1-245 ) Delivery by YopE 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 Type I IFN induction was evaluated in B16F1 melanoma, RAW macrophages, and human THP-1 reporter cell lines in relation to the copy number of vectors encoding both (Figure 16). The B16F1 reporter cell line can be used primarily to evaluate RIG1-dependent signaling, the THP-1 cell line can be used primarily to evaluate cGAS-dependent signaling, and the RAW cell line can be used to evaluate the potential for co-activation of cGAS and RIG-1 (Figure 18). Reporter cells were induced in YopE 1-138 - Human RIG-1CARD2 and YopE 1-138 - Human cGAS 161-522 The bacteria were infected with strains expressing and transferring various amounts (MOI) of YopE. 1-138 - Human cGAS 161-522 YopE combined with 1-138 Human RIG-1 CARD2 demonstrated dose-dependent induction of a type I IFN response in all reporter cell lines (Figure 16), but the bacterial background strain (Y. enterocolitica ΔHOPEMT) was unable to induce such a response (Figure 16).

[0262] In all cell lines tested, YopE 1-138 - Human RIG-1CARD2 and YopE 1-138 - Human cGAS 161-522 pYV encoding and YopE 1-138 - Human RIG-1CARD2 and YopE 1-138 - Human cGAS 161-522The combination of vector-based encoding induced the highest type I IFN activation when the vector was a medium copy number vector (Figure 16), followed by low copy number, and weakest with high copy number plasmids (Figure 16). The results indicate that dual encoding on pYV and the vector is beneficial for increased type I IFN activation (see Figures 10 and 19) and genetic stability (see Figures 20 and 21), but these data suggest that medium copy number vectors are optimal.

[0263] YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 ), YopE 1-138 - Human cGAS 161-522 Or YopE 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 To summarize the results regarding the effect of the copy number of the vector encoding (Figures 14-16), a medium copy number vector is the most preferable.

[0264] Here, YopE 1-138 - Human RIG-1 CARD2 (RIG-I 1-245 ) and YopE 1-138 - Human cGAS 161-522 Figure 17 shows the optimal combination for delivery of two proteins encoded by endogenous pathogenic and medium copy number vectors, as shown for human cGAS. 161-522 Furthermore, the optimized delivery and genetic stability of RIG-I CARD2 are achieved by encoding on an endogenous pYV plasmid and, additionally, on a medium copy number vector.

[0265] Type I IFN-induced protein YopE 1-138 - Human cGAS 161-522 and YopE 1-138 By studying the delivery of RIG-I CARD2 and its effects on different cell types, we evaluated the difference in inductive ability between these two proteins. Optimized combinations of fusion proteins should enable activation of a diverse set of cell types. YopE 1-138- Human cGAS 161-522 and YopE 1-138 - Delivery of RIG-I CARD2 results in a difference in the induction of type I IFN signaling in B16F1 melanocytes (Figure 18A), human glioblastoma LN-229 (Figure 18B), mouse RAW macrophages (Figure 18C), or human THP-1 macrophages (Figure 18D). YopE 1-138 -RIG-I CARD2 is a mouse B16F1 and human LN-229 glioblastoma cell, and YopE 1-138 - Human cGAS 161-522 This results in stronger type I IFN induction. In mouse raw macrophages, YopE 1-138 - Human cGAS 161-522 and YopE 1-138 -RIG-I CARD2 has equivalent type I IFN induction ability, but YopE 1-138 - Human cGAS 161-522 This is a human THP-1 macrophage and YopE 1-138 - It is superior to the RIG-I CARD2.

[0266] As an example, the A20 mouse B-cell lymphoma cell line is Y. enterocolitica ΔHOPEMT, and YopE is placed on a medium copy number plasmid. 1-138 - Encodes human RIG-I CARD2 and places YopE on a medium copy number plasmid. 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 Encodes YopE on medium copy number plasmids and pYV 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 It was infected with something that codes for YopE on a medium copy number plasmid. 1-138 -The strain encoding human RIG-I CARD2 has YopE on a medium copy number plasmid. 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 It induces less type I IFN response in A20 cells than strains encoding YopE, and this also applies to medium copy number plasmids and pYV. 1-138- Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 The strains encoding the specified character are below the threshold (Figure 19A).

[0267] In another example, the Jurkat human T cell line was modified with Y. enterocolitica ΔHOPEMT, and YopE was added to a medium copy number plasmid. 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 Encodes YopE on medium copy number plasmids and pYV 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 It was infected with something that codes for YopE on a medium copy number plasmid. 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 The strain encoding YopE on the medium copy number plasmid and pYV 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 The strains encoding the specified character are below the threshold (Figure 19B).

[0268] To evaluate the genetic stability of a bacterial strain when it is growing in vivo (growing within a tumor), mice carrying syngeneic subcutaneous B16F10 melanoma tumors were given YopE on an endogenous pYV plasmid. 1-138 - Human cGAS 161-522 and YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 The Y. enterocolitica subspecies paleacutica MRS40 ΔyopH, O, P, E, M, and T strains, which encode both of these, were colonized by intravenous administration, and bacteria isolated from the tumors were evaluated for the presence of each select marker. When the subcutaneous tumor reached a predetermined size (approximately 100-200 mm3), the mice were subjected to YopE fertilization onto the endogenous pYV plasmid. 1-138 - Human cGAS 161-522 and YopE 1-138 - Encoding both sides of Human RIG-I CARD2, 1×10 7Mice were intravenously infected with cfu strains of Y. enterocolitica subspecies Palaearctica MRS40 ΔyopH,O,P,E,M,T. To enable bacterial growth, mice were pre-treated with desfreoxamine 24 hours prior to infection. Mice infected with the attenuated strains of Y. enterocolitica subspecies Palaearctica MRS40 ΔyopH,O,P,E,M,T showed no significant weight loss and were scored normally for physical appearance and behavior. Bacteria were isolated on days 2 and 4 post-infection, and replicas were collected on selective agar plates to assess the presence of endogenous pYV plasmids (Figure 20). In almost all animals evaluated, the pYV plasmid was found to be highly stable and present on days 2 and 4 post-infection (Figure 20).

[0269] Similarly, in mice carrying syngeneic subcutaneous EMT-6 mammary tumors, YopE was added to the endogenous pYV plasmid. 1-138 - Human cGAS 161-522 and YopE 1-138 - Human RIG-I CARD2 (RIG-I 1-245 The Y. enterocolitica subspecies paleacutica MRS40 ΔyopH, O, P, E, M, and T strains, which encode both , were colonized by intratumoral administration, and bacteria isolated from the tumors were evaluated for the presence of each selective marker. When the subcutaneous tumor reached a predetermined size (approximately 100-200 mm3), mice were subjected to a procedure to enlarge the endogenous pYV plasmid to YopE 1-138 - Human cGAS 161-522 and YopE 1-138 - Encoding both sides of Human RIG-I CARD2, 7.5 × 10 7Mice were intravenously infected with cfu strains of Y. enterocolitica subspecies palearchica MRS40 ΔyopH, O, P, E, M, T. To enable bacterial growth, mice were pre-treated with desfreoxamine before infection. Mice infected with the attenuated strains of Y. enterocolitica subspecies palearchica MRS40 ΔyopH, O, P, E, M, T showed no significant weight loss and were scored normally for physical appearance and behavior. Bacteria were isolated on post-infection days 1 and 2, and replicas were collected on selective agar plates to assess the presence of endogenous pYV plasmid and medium copy number vector (Figure 21). In almost all animals evaluated, the pYV plasmid and vector were found to be highly stable and present on post-infection days 1 and 2 (Figure 21). Some isolated bacterial colonies contained only the pYV plasmid and lacked the medium copy number vector, and vice versa. Overall, the pYV plasmid was found to be slightly more stable than the medium copy number vector up to day 2 after administration.

[0270] YopE delivered to tumor cells in vivo 1-138 - Human RIG1 CARD2 (RIG-I 1-245 ) and YopE 1-138 - Human cGAS 161-522 To evaluate the effects, we conducted a study in wild-type C57BL / 6 mice that had been subcutaneously allografted with B16F10 melanoma cells. When the tumors reached a size of approximately 60–130 mm³, the mice were given PBS (Figure 23) or 7.5 × 10⁶ 7 YopE encoded in Y. enterocolitica ΔHOPEMT (Figure 24) and Y. enterocolitica ΔHOPEMT + endogenous pYV plasmid (endogenous sites of yopH and yopE, respectively). 1-138 Human RIG1 CARD2 and YopE 1-138 Human cGAS 161-522 (Figure 25), and additionally, those encoded in medium copy number vectors (YopE 1-138 - Human cGAS 161-522 and YopE 1-138-RIG-I CARD2 (encoded into a single operon under the control of the yopE promoter) was injected intratumorally (it). The day of the first intratumor injection of the bacteria was defined as day 0. Mice were injected intratumorally on d0, d1, d5, d6, d10, and d11. Tumor volume was measured with calipers over the following days. Treatment with Y. enterocolitica ΔHOPEMT alone affected the progression of tumor volume, with 2 / 15 mice showing complete tumor regression (Figure 24). YopE 1-138 - Human cGAS 161-522 and YopE 1-138 - Y. enterocolitica ΔHOPEMT delivering RIG-I CARD2 was found to have a more significant effect on tumor progression, with 8 / 15 mice showing complete and sustained tumor regression (Figure 25). Furthermore, the mean tumor volume was compared between Y. enterocolitica ΔHOPEMT alone and Y. enterocolitica ΔHOPEMT combined with YopE 1-138 - Human cGAS 161-522 and YopE 1-138 - The effects of RIG-I CARD2 delivery are evident (Figure 22). These findings highlight that such bacteria and their T3SS can be used for highly significant inhibition of tumor progression, and that the delivery of type I IFN-inducing proteins is suitable for inducing regression of primary tumors.

[0271] In summary, YopE coded into a vector 1-138 - Human cGAS 161-522 And, to a smaller degree, YopE is coded into a vector. 1-138 - Human RIG-I CARD2 increases the activation of type I IFN response more than pYV plasmid encoding (Figures 8 and 9). YopE on the vector 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522Dual encoding does not impair the activity of each individual cargo (Figures 11 and 12). Encoding two different heterologous cargoes has the advantage of potentially broader activation across diverse cell types (Figures 18 and 19). Therefore, while vector-based encoding appears necessary for high activation potential, dual encoding of two heterologous cargoes was found to increase overall activity, namely RIG-I CARD2 and cGAS 161-522 The combination is cGAS 161-522 Alternatively, it led to interferon signaling in a wider variety of cell types compared to delivery of RIG-I CARD2 alone.

[0272] pYV encoding of heterologous proteins has the advantage of increased in vivo genetic stability compared to vector-based encoding (Figures 20 and 21). YopE 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 The pYV and vector-based encodings were found to be superior to or equivalent to vector-based encodings alone in terms of the potential for IFN activation (Figures 10 and 19). Overall, these findings indicate that combined delivery of human cGAS and human RIG-I CARD increases the range of cell types that can achieve type I IFN induction (compared to delivery of cGAS alone or RIG-I CARD alone) and enhances the potency of interferon induction in several cell types.

[0273] Therefore, YopE 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522 The dual encoding of pYV and vectors is done by YopE 1-138 - Human RIG-I CARD2 and YopE 1-138 - Human cGAS 161-522It can be considered optimal for maximum activity (see Figure 19 for example) and offers the best genetic stability (see Figures 20 and 21). In addition, medium copy number vectors were found to be superior to low copy number or high copy number vectors (Figures 13-16). (For example, as shown in Figure 17) YopE can be placed on medium copy number vectors. 1-138 - Human cGAS 161-522 and YopE 1-138 -RIG-I CARD2 is coded into a single operon, YopE 1-138 - Human cGAS 161-522 and YopE 1-138 A strain combining pYV and medium copy number vector-based encoding of RIG-I CARD2 combines all the beneficial features (potential for broad cell type activation, increased potency, and genetic stability). Such a strain (shown in Figure 17) was ultimately validated for its impact on tumor progression in animal models of solid tumors (Figures 22-25), and was found to produce persistent and complete tumor regression in over 50% of the treated animals.

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Claims

1. This is a recombinant, attenuated strain of Yersinia enterocolitica, a recombinant Gram-negative bacterial strain. i) A first polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; ii) A second polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is ligated to a promoter in a manner that allows it to act; iii) A third polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act; and iv) A fourth polynucleotide molecule comprising a nucleotide sequence encoding a heterologous protein or fragment thereof, which is in-frame fused to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding the delivery signal from a bacterial effector protein is ligated to a promoter in a manner that allows it to act. A recombinant Gram-negative bacterial strain comprising the first and second polynucleotide molecules located on a vector contained in the Gram-negative bacterial strain, and the third and fourth polynucleotide molecules located on the chromosome of the Gram-negative bacterial strain or on an extrachromosomal genetic element contained in the Gram-negative bacterial strain, wherein the extrachromosomal genetic element is not the vector on which the first and second polynucleotide molecules are located. The nucleotide sequence encoding a heterogeneous protein or fragment thereof in the first polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof in the third polynucleotide molecule encode the same heterogeneous protein or fragment thereof. The nucleotide sequence encoding a heterogeneous protein or fragment thereof in the second polynucleotide molecule and the nucleotide sequence encoding a heterogeneous protein or fragment thereof in the fourth polynucleotide molecule encode the same heterogeneous protein or fragment thereof. The heterologous proteins or fragments thereof encoded by the first and third polynucleotide molecules are different from the heterologous proteins or fragments thereof encoded by the second and fourth polynucleotide molecules. Fragments of heterologous proteins contain between 100 and 800 amino acids and possess the same functional properties as the heterologous protein from which they originate. The delivery signal from the bacterial effector protein is the bacterial T3SS effector protein or its N-terminal fragment. The N-terminal fragment of the bacterial T3SS effector protein contains at least the first 10 amino acids of the bacterial T3SS effector protein. The bacterial T3SS effector protein or its N-terminal fragment contains a chaperone-binding site, and The heterologous proteins or fragments thereof encoded by the nucleotide sequences of the first, second, third, and fourth polynucleotide molecules are independent of each other: STING, TRIF, TBK1, IKKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1, MDA5, LGP2, MAVS, Trim25, Trim32, Trim56, Riplet, TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF 7. Proteins involved in the induction or regulation of type I IFN responses, selected from the group consisting of IRF9, STAT1, STAT2, PKR, TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSm14A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, CRADD, RIPK2, CARD6, NOD1, NOD2, WspR, DncV, DisA, CdaA, CdaS, and cGAS; pro-apoptotic proteins and anti-apoptotic proteins Proteins involved in apoptosis, selected from the group consisting of proteins; cell cycle regulators, selected from the group consisting of cyclins, cyclin-dependent kinases (CDKs), CDK-activated kinases, CDK inhibitors, CDK substrates, and cell cycle checkpoint proteins; ankyrin repeat proteins; cell signaling proteins, selected from the group consisting of cytokine signaling proteins, survival factor signaling proteins, death signaling proteins, growth factor signaling proteins, hormone signaling proteins, chemokine signaling proteins, and hedgehog signaling proteins; reporter proteins, selected from the group consisting of fluorescent proteins, luciferases, and enzyme reporter proteins; transcription factors; proteases; low molecular weight GTPases; GPCR-related proteins, selected from the group consisting of G protein-coupled receptors, G protein complexes composed of G alpha, G beta, and G gamma, kinases, adapter proteins, signaling factors, and signaling regulators;Nanobody fusion constructs selected from the group consisting of nanobodies fused to proteolytic domains selected from the group consisting of F-box domains and ubiquitin ligases, nanobodies fused to cell signaling proteins, bispecific or multispecific nanobodies, nanobodies fused to reporter proteins, and nanobodies fused to intracellular localization signals; nanobodies; bacterial T3SS effector proteins; bacterial T4SS effector proteins and viral proteins; and fragments of these heterologous proteins selected from the group consisting of these heterologous protein fragments containing between 100 and 800 amino acids and having the same functional properties as the heterologous protein from which they are derived; Recombinant Gram-negative bacterial strain.

2. A recombinant Gram-negative bacterial strain according to claim 1, wherein a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a first polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a second polynucleotide molecule, are linked in such a way as to act on the same promoter.

3. A recombinant Gram-negative bacterial strain according to claim 1 or 2, wherein a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a third polynucleotide molecule, and a nucleotide sequence encoding a heterologous protein or fragment thereof, fused in-frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of a fourth polynucleotide molecule, are linked in a manner that can act on two different promoters.

4. A recombinant Gram-negative bacterial strain according to any one of claims 1 to 3, wherein the vector is a medium copy number plasmid.

5. A recombinant Gram-negative bacterial strain according to any one of claims 1 to 4, wherein the extrachromosomal genetic element is an endogenous pathogenic plasmid.

6. A recombinant Gram-negative strain according to any one of claims 1 to 5, wherein heterologous proteins or fragments thereof encoded by the nucleotide sequences of the first, second, third, and fourth polynucleotide molecules are independently selected from the group consisting of WspR, DncV, DisA, CdaA, CdaS, and cGAS, and fragments thereof, which lead to stimulation of RIG1, MAVS, CRADD, RIPK2, CARD6, NOD1, NOD2, and STING.

7. The recombinant Gram-negative bacterial strain according to any one of claims 1 to 6, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the first and third polynucleotide molecules is a cGAS.

8. A recombinant Gram-negative bacterial strain according to any one of claims 1 to 6, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the first and third polynucleotide molecules is a cGAS fragment, and the cGAS fragment contains amino acids between 100 and 800 and has the same functional properties as cGAS.

9. The recombinant Gram-negative strain according to any one of claims 1 to 8, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the first and third polynucleotide molecules is a fragment of cGAS shown in Sequence ID No.

10.

10. The recombinant Gram-negative bacterial strain according to any one of claims 1 to 9, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the second and fourth polynucleotide molecules is RIG1.

11. The recombinant Gram-negative bacterial strain according to claim 1, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the second and fourth polynucleotide molecules is a fragment of RIG1, and the fragment of RIG1 includes a CARD domain, includes amino acids between 100 and 800, and has the same functional properties as RIG1 including the CARD domain.

12. The recombinant Gram-negative bacterial strain according to claim 1, wherein the heterologous protein or fragment thereof encoded by the nucleotide sequences of the second and fourth polynucleotide molecules is a fragment of RIG1 shown in SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO:

3.

13. A recombinant Gram-negative bacterial strain according to any one of claims 1 to 12, for use as a pharmaceutical.

14. A recombinant gram-negative strain according to any one of claims 1 to 12, for use in a method for treating a target cancer, wherein the method comprises administering the recombinant gram-negative strain to the target, and the recombinant gram-negative strain is administered in an amount sufficient to treat the target.

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