EA044058B1 - DELIVERY OF PROTEINS BASED ON VIRULENCE-ATTENUATED BACTERIA - Google Patents

Description

Field of technology

The invention relates to recombinant virulence-attenuated gram-negative strains of bacteria and their use in a method for treating cancer in a subject.

State of the art

Bacteria use various mechanisms to directly inject proteins into target cells ¹ . The type III secretion system (T3SS), used by bacteria such as Yersinia, Shigella and Salmonella ² , functions like a nanosyringe that injects so-called bacterial effector proteins into host cells.

T3S has been used to deliver hybrid peptides and proteins to target cells. Heterologous bacterial T3SS effectors were delivered when the bacterium of interest was refractory to genetic targeting (such as Chlamydia trachomatis). Often, reporter proteins were fused to putative T3SS secretion signals depending on the study requirements for T3SS-dependent delivery of proteins such as Bordetella pertussis adenylate cyclase, mouse DHFR (dihydrofolate reductase), or a phosphorylatable tag. Peptide delivery has mainly been carried out for the purpose of vaccination. Peptides included viral epitopes, bacterial epitopes (listeriolysin O), and peptides representing human cancer cell epitopes. In several cases, functional eukaryotic proteins have been delivered for host cell modulation, which has been accomplished using nanobodies ³ , nuclear proteins (Cre recombinase, MyoD) ⁴⁵ or Il10 and IL1ra ⁶ . None of the above systems allows delivery of single proteins, since in each case one or multiple endogenous effector proteins are encoded. Moreover, the vectors used were constructed in a manner that does not allow easy cloning of other DNA fragments encoding preferred proteins, preventing widespread use of the system.

Approaches that provide targeted drug delivery are of great interest. For example, antibodies are used that recognize the surface structures of tumor cells and, optimally, selectively bind to tumor cells. To improve the mechanism of action of such antibodies, they can be conjugated to therapeutic agents or to lipid vesicles loaded with drugs. One of the problems with the use of such vesicles is the proper release of the active component. Even more challenging is the delivery of therapeutic proteins or peptides, particularly when targeting intracellular mechanisms. Many alternative methods have been tried to solve this problem of delivering therapeutic proteins to eukaryotic cells, which include cell penetrating peptides (CPP) or similar technologies, as well as various nanoparticle-based methods. All of these technologies have the disadvantages of low efficiency and the fact that cargo taken up by the cell through endocytosis is likely to eventually be broken down in lysosomes. Moreover, the conflict between the need to ensure stability of the cargo transporter in the human body and the need to destabilize and release it in the target cell is a problem inherent in such technologies.

A variety of bacteria have been shown to replicate in malignant solid tumors when introduced from a distant site, including Escherichia coli, Vibrio cholerae, Salmonella enterica, Listeria monocytogenes, Pseudomonas aeruginosa, and Bifidobacteria. To date, only Bacillus Calmette-Guerin (BCG, derived from Mycobacterium bovis) is used in clinical practice. BCG is administered to treat superficial bladder cancer, although the underlying molecular mechanism remains largely unknown. The development of strains of bacteria that are capable, for example, of delivering a cargo formed inside the bacterium to its site of action inside cells, such as cancer cells, i.e. outside of bacteria remains an important task.

Brief description of the invention

The present invention relates to recombinant virulence-attenuated Gram-negative bacterial strains and their use in a method of treating cancer in a subject. In some embodiments, the present invention provides recombinant virulence-attenuated Gram-negative bacterial strains and their use for treating cancer in a subject, wherein the recombinant virulence-attenuated Gram-negative bacterial strains provide translocation of various type III effectors, as well as type IV effectors, viral proteins, and that most importantly, functional eukaryotic proteins into cancer cells, such as malignant solid tumor cells. The present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain with enhanced heterologous protein expression and secretion properties, which is surprisingly capable of stably encoding a heterologous protein for several days or even weeks in vivo.

In a first aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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 wherein a nucleotide sequence encoding a delivery signal from a bacterial effector protein is operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing the nucleotide sequence, which contains a gene encoding the specified endogenous protein necessary for growth, operably linked to the promoter.

In a further aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, is operatively linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain additionally contains modulation in the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type.

In a further aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, is operably linked to a promoter, and wherein the heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response.

In a further aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence, encoding a delivery signal from a bacterial effector protein, is operatively linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding said endogenous protein required for growth operably linked to a promoter, for use in a method of treating cancer in a subject, wherein said method comprises administering to the subject said recombinant virulence-attenuated Gram-negative strain of bacteria, wherein the recombinant virulence-attenuated Gram-negative strain of bacteria is administered in an amount that is sufficient to treat the subject. Similarly, the present invention relates to a method of treating cancer in a subject, comprising administering to the subject a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is operably linked to the promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid, containing a nucleotide sequence that contains a gene encoding said endogenous protein required for growth, operably linked to a promoter, wherein the recombinant virulence-attenuated Gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject.

Similarly, the present invention relates to the use of a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, is operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains the gene, encoding said endogenous protein required for growth, operably linked to a promoter, for the purpose of producing a medicament for the treatment of cancer in a subject.

In a further aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to

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the 3'-end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding a delivery signal from a bacterial effector protein is operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further comprises modulation in the temperature-sensitive region of the RNA in the direction 3'-5' from a gene encoding an endogenous DNA-binding protein of the AraC type, for use in a method of treating cancer in a subject, the method comprising administering to the subject said recombinant virulence-attenuated Gram-negative strain of bacteria, wherein the recombinant virulence-attenuated Gram-negative strain of bacteria is administered in an amount , which is sufficient to treat the subject.

Similarly, the present invention relates to a method of treating cancer in a subject, comprising administering to the subject a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence encoding the delivery signal from the bacterial effector protein is operatively linked to the promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain additionally contains modulation in the thermosensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous A DNA binding protein of the AraC type, wherein the recombinant virulence-attenuated Gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject. Similarly, the present invention relates to the use of a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to the 3'-terminus of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, is functionally linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain additionally contains modulation in the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type with the purpose obtaining a medicament for treating cancer from a subject.

In a further aspect, the present invention relates to a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to the 3'-terminus of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, operably linked to a promoter, and wherein the heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response, for use in a method of treating cancer in a subject, wherein said method includes administering to the subject said recombinant a virulence-attenuated gram-negative bacterial strain, wherein the recombinant virulence-attenuated gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject.

Similarly, the present invention relates to a method of treating cancer in a subject, comprising administering to the subject a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains 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, wherein the nucleotide sequence encoding a delivery signal from the bacterial effector protein is operably linked to a promoter, and wherein the heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response, wherein the recombinant virulence-attenuated Gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject.

Similarly, the present invention relates to the use of a recombinant virulence-attenuated Gram-negative bacterial strain containing a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to the 3' terminus of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, the nucleotide sequence encoding a delivery signal from a bacterial effector protein, operably linked to a promoter, and wherein the heterologous protein is a protein involved in inducing or regulating an interferon (IFN) response for the purpose of producing a drug for treating cancer in a subject.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a recombinant virulence-attenuated Gram-negative bacterial strain and a pharmaceutically acceptable carrier, wherein the recombinant virulence-attenuated

- Z 044058 gram-negative bacterial strain contains a nucleotide molecule that contains 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, wherein the nucleotide sequence encoding a delivery signal from a bacterial effector protein, functionally linked to the promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding said endogenous protein necessary for growth, functionally linked to the promoter. In a further aspect, the present invention relates to a pharmaceutical composition comprising a recombinant virulence-attenuated gram-negative bacterial strain and a pharmaceutically acceptable carrier, wherein the recombinant virulence-attenuated gram-negative bacterial strain contains a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to a 3' - the end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding a delivery signal from a bacterial effector protein is operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further comprises modulation in the temperature-sensitive region of the RNA in the 3' direction -5' from the gene encoding the endogenous DNA-binding protein of the AraC type.

In a further aspect, the present invention relates to a pharmaceutical composition comprising a recombinant virulence-attenuated gram-negative bacterial strain and a pharmaceutically acceptable carrier, wherein the recombinant virulence-attenuated gram-negative bacterial strain contains a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame to a 3' -the end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein, wherein the nucleotide sequence encoding a delivery signal from a bacterial effector protein is operably linked to a promoter, and wherein the heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response.

Brief description of the figures

Fig. 1 - Characteristics of protein delivery using T3SS. Schematic representation of T3SS-dependent protein secretion into the environment (in vitro secretion) (left panel) or into eukaryotic cells (right panel). I: type 3 secretion system is shown. II: proteins secreted into the environment are shown, III - proteins translocated through the membrane into the cytosol of eukaryotic cells (VII). VI: Shows the region of two bacterial membranes in which the T3SS is embedded and the underlying bacterial cytosol. IV is a fusion protein attached to the N-terminal fragment of YopE ₁ - ₁₃₈ (V).

Fig. 2 is a description of the type III secretion based delivery toolkit. ₍ A) Vector maps of the pBad_Si1 and pBad_Si2 cloning plasmids that were used to generate YopE _{1_138} fusion constructs. The SycE chaperone and the YopE _{1_138} fusion are under the control _of the native Y. enterocolitica promoter. The two plasmids differ solely in the presence of arabinose-inducible EGFP (enhanced green fluorescent protein) present on pBad_Si1 (B) The multiple cloning site is located immediately after the yopE1_ ₁₃₈ fragment on plasmids pBad_Si1 and pBad_Si2.

Fig. ZA-q - strains of Y. enterocolitica used in this study. The list of Y. enterocolitica strains used in this study provides information on the background strains, plasmids, and proteins for T3SS-dependent delivery on the corresponding plasmids. Information is also provided on the oligonucleotides used to construct the corresponding plasmid, the scaffold plasmid, and antibiotic resistance.

Fig. 4 - virulence plasmid of Yersinia enterocolitica W227, pYV. Yersinia strain W227 virulence plasmid (pYV) size 69673 bp. presented to scale. Effector proteins T3SS, origin of replication and arsenic resistance (encoded by the arsC, B, R and H genes) are marked: 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-XVXX: arsC, B, R and H.

Fig. 5 - delivery of synthetic increased proapoptotic proteins. Delivery of single synthetic proteins consisting of single or tandem repeats of BH3 domains derived from the proapoptotic proteins t-BID or BAX leads to enhanced induction of apoptosis in 4T1 and B16F10 cancer cells. 4T1 (I) or B16F10 (II) cells were infected with Y. enterocolitica AyopHOPEMT encoded on pBad-MycHisA IV: YopE _1-138 - extended domain BH3 tBID, V: YopE ₁ . ₁₃₈ -linker-BH3 tBID, VI: YopE1-138-BH3 tBID, VII: YopE _M 38-(BH3 tBID) ₃ , VIII: YopE1-138-BH3 tBID - BH3 CVC or IX: YopE1i38-BH3 BAX - BH3 tBID. Titration of bacteria added to cells (multiplicity of infection, MOI) was performed for each strain, cell number was determined, and IC ₅₀ was calculated using nonlinear regression. MZ IC ₅₀ (III) is indicated.

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Fig. 6 - induction of apoptosis by synthetic proapoptotic proteins encoded by pYV. Delivery of single or tandem repeat domain BH3 BID encoded on pYV results in induction of apoptosis in 4T1 and B16F10 cancer cells. 4T1 (I) or B16F10 (II) cells were infected with Y. enterocolitica AHOPEMAT+IV: pYV-YopE ₁ . ₁₃₈ -BH3-Bid or V:+pYV-YopE ₁ . ₁₃₈ -(BH3-Bid)2 or VI: using Y. enterocolitica AHOPEMAT pBad-MycHisA-YopE ₁ _ ₁₃₈ -(BH3-Bid) ₂ for 3 hours. Titration of bacteria added to cells (MH) was carried out for each strain, cell number was determined and IC50 (III) was calculated using nonlinear regression.

Fig. 7 - colonization of the tumor by injected i.v. (intravenous) Y. enterocolitica AyopH,O,P,E,M,T in a 4T1 breast cancer allograft model. The number of bacteria in tumors is reported as colony-forming units (CFU) per g of tissue (III). Counting was carried out in tumors on days 8 (I) and 14 (II) after infection. Each dot represents an individual mouse. The horizontal dotted line represents the detection limit.

Fig. 8 - biodistribution of injected i.v. Y. enterocolitica AuopH,O,P,E,M,T in the 4T1 breast cancer allograft model. The number of bacteria in the blood (I), spleen (II), liver (III), lung (IV) and tumor (V) is indicated as colony-forming units (CFU) per 1 g of tissue or per 1 ml of blood (VI). Counting was performed on day 14 postinfection. Each dot represents an individual mouse. The horizontal dotted line represents the detection limit. * denotes a mouse with extensive metastases found in the lungs.

Fig. 9 - delay in tumor progression in wild-type Balb/C mice with s.c. (subcutaneous) allograft of 4T1 breast cancer cells. Wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells i.v. injected I: PBS (phosphate buffer solution) or II: 1χ 10 ⁷ Y. enterocolitica dHOPEMT AHairpinI-VirF+pYV-YopE1 _-138 (BH3-Bid)2 when the tumor reached a size of 150-250 mm ³ . Day v.v. injection of bacteria was designated as day 0. Tumor volume was measured during the following days (III; day 0 to day 9 after i.v. injection of bacteria) using a caliper. The relative tumor volume normalized to the tumor volume at day 0 is indicated (IV) in mm ³ . The mean is indicated by symbols, and the error bars shown show the standard error of the mean. Statistical significance was measured using two-way ANOVA, * indicates p-value <0.05, ** indicates p-value <0.005.

Fig. 10 - tumor progression in wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells. Wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells i.v. injected I: PBS or II: 1x10 ⁷ Y. enterocolitica dHOPEMT when the tumor reached a size of 150-250 mm ³ . Day v.v. injection of bacteria was designated as day 0. Tumor volume was measured during the following days (III; day 0 to day 9 after i.v. injection of bacteria) using a caliper. The relative tumor volume normalized to the tumor volume at day 0 is indicated (IV) in mm ³ . The mean is indicated by symbols, and the error bars shown show the standard error of the mean.

Fig. 11 - regulation of secretion based on T3SS through control of the expression of the master regulator VirF. A: In vitro secretion assay (performed at 37°C) with Y. enterocolitica AHOPEMAT strains delivering YopE1_ ₁₃₈ -(BH3 tBID) ₂ . Expression of VirF is under the control of its native promoter (I+II), its arabinose-inducible promoter (III+IV), or its native promoter with deletion of the hairpin I region controlling temperature-dependent expression (V). The secretion assay was performed in the absence of arabinose (I, III and V) or in the presence of 0.2% arabinose (II and IV). Secreted YopE1_ ₁₃₈ -(BH3 tBID) ₂ was detected using Western blotting with an antibody recognizing the YopE1_ ₁₃₈ region. B: In vitro secretion assay (performed at 37°C) with YopE ₁ -delivering Y. enterocolitica AHOPEMAT strains. ₁₃₈ -domains of mouse Card RIG1. Expression of VirF is under the control of its native promoter (I+II) or its native promoter with deletion of the hairpin I region controlling temperature-dependent expression (III). The secretion assay was performed in the absence of arabinose (I and III) or in the presence of 0.2% arabinose (II). Secreted YopE ₁ . The ₁₃₈ -domains of mouse Card RIG1 were detected using Western blotting with an antibody recognizing the YopE _1-138 region.

Fig. 12 - comparison of in vitro growth. Comparison of in vitro growth for II: Y. enterocolitica AHOPEMAT, III: Y. enterocolitica AHOPEMAT Aasd, IV: Y. Enterocolitica AHOPEMT Aasd+pBAD-MycHisA-asd, V: Y. enterocolitica AHOPEMAT Aasd+pBAD-MycHisA-asd (reverse orientation ), VI: Y. enterocolitica AHOPEMAT, encoding YopE1_ ₁₃₈ -(BH3 tBID)2 on pYV and VII: Y. enterocolitica AHOPEMAT Aasd+pYV-asd-YopE1. ₁₃₈ -(BH3 tBID) ₂ . Bacteria were inoculated in liquid culture and grown for 3 hours. OD600 (optical density at 600 nm) was then determined for all strains (I).

Fig. 13 - tumor colonization by Y. enterocolitica AHOPEMAT Aasd+pBad-MycHisA-asd and stability of pBad-MycHisA-asd: Wild-type C57BL/6 mice with s.c. allograft of melanoma cells B16F1O i.v. injected with 1x106 Y. enterocolitica AHOPEMAT Aasd+pBad-MycHisA-asd. On day 1 (I) or day 4 (II) after i.v. bacterial injections, blood (V), spleen (VI), liver (VII), lung (VIII) and tumor (IX) were collected, homogenized, serial diluted and plated on LB agar plates containing nalidixic acid (and free of ampicillin, -IV), or on LB agar plates with ampicillin (+IV), selective for pBad-MycHisA-asd. The number of bacteria in the corresponding samples is reported as colony-forming units (CFU) per gram of tissue or ml of blood (III). Each dot represents an individual mouse. The horizontal dotted line represents the detection limit.

Fig. 14 - tumor colonization with Y. enterocolitica AHOPEMAT Aasd+pBad-MycHisA-asd: Wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells. V. injected with 1x10 ⁶ Y. enterocolitica AHOPEMAT Aasd+pBad-MycHisA-asd. On the indicated days after i.v. injection of bacteria (I) tumors were selected, homogenized, serial dilutions were prepared, and plated on LB agar plates containing nalidixic acid. The number of bacteria in tumors is reported as colony-forming units (CFU) per g of tissue (II). Each dot represents an individual mouse. The horizontal dotted line represents the detection limit.

Fig. 15 - genetic stability of pYV: Stability of native pYV or pYV-asd in solid tumors in vivo. Wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells i.v. injected 1χ10 ⁷ II: Y. enterocolitica AHOPEMAT+pYV-YopE1. ₁₃₈ -(BH3 tBID)2, III: Y. enterocolitica AHOPEMAT AhaiipmI-virF+pYV-YopE1. ₁₃₈ -(BH3 tBID)2 or IV: Y. enterocolitica AHOPEMAT Aasd+pYV-asd-YopE1. ₁₃₈ -(BH3 tBID)2. On day 9 after i.v. injections of tumor bacteria were selected, homogenized, serial dilutions were prepared, and plated on LB agar plates containing nalidixic acid. After growth on these plates, single colonies from individual mice were transferred to LB agar plates with and without sodium arsenite, selective for pYV. For each mouse, the percentage of colonies that grew on agar plates containing arsenite to the number of colonies that grew on plates not containing arsenite is indicated (I: as a %). 100% means that all colonies isolated from a solid tumor still contain the pYV plasmid.

Fig. 16 - tumor colonization. Wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells i.v. injected 1χ10 ⁷ II: Y. enterocolitica AHOPEMAT+pYV-YopE1. ₁₃₈ -(BH3 tBID)2, III: Y. enterocolitica AHOPEMAT AhairpinI-virF+pYV-YopE1. ₁₃₈ -(BH3 tBID)2 or IV: Y. enterocolitica AHOPEMAT Aasd+pYV-asd-YopE1. ₁₃₈ -(BH3 tBID)2. On day 9 after i.v. injections of tumor bacteria were selected, homogenized, serial dilutions were prepared, and plated on LB agar plates containing nalidixic acid. The number of bacteria in tumors is reported as colony-forming units (CFU) per g of tissue (I). Each dot represents an individual mouse. The horizontal dotted line represents the detection limit.

Fig. 17 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - Rig1 pathway. Delivery of human and mouse Card Rig1 domains results in the induction of type I IFN in the IFN reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on a pBadMycHisA-derived plasmid II: YopEi.i3 ₈ - human Card Rig1 domains, III: YopE ₁ . ₁₃₈ -domains of mouse Card Rig1. Titration of bacteria added to the cells (IV: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (V: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54, enhanced by the multimeric ISRE (interferon-stimulated responsive element, interferon-stimulated responsive element).

Fig. 18 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - Rig1 pathway. Delivery of human Card Rig1 domains results in the induction of type I IFN in the IFN reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE1_ ₁₃₈ -MycHis, III: YopE1_ ₁₃₈ - human Card Rig1 domain. Titration of bacteria added to the cells (IV: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (V: OP650), which is under the control of the IISG54 promoter, consisting of the IFN-inducible ISG54 promoter. enhanced by a multimeric ISRE.

Fig. 19 - delivery of proteins that induce a type I interferon response using bacterial T3SS - Rig1 pathway: positive control. Positive control in the same experiment as in FIG. 18, using IFN gamma to stimulate the IFN reporter cell line B16F10. B16F10 reporter cells were stimulated with mouse IFN gamma. IFN gamma titer (I: indicated in U/ml) was added to the cells, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (II: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter, enhanced by a multimeric ISRE.

Fig. 20 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - Rig1 pathway. Delivery of pYV-encoded domains of mouse Card Rig1 results in the induction of type I IFN in the B16F10 cancer cell line. B16F10 cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on pYV II: YopE ₁ . ₁₃₈ -domains of mouse Card Rig1. Titration of bacteria added to the cells (III: indicated in the MS) was performed for each strain, and IFN stimulation was assessed by adding cell supernatant to an IFN reporter cell line based on the activity of secreted alkaline phosphatase (IV: OP650), which is found under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

Fig. 21 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - Rig1 pathway. Delivery of pYV-encoded Card Rig1 domains to mice results in the induction of type I IFN in the 4T1 cancer cell line. 4T1 cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on pYV II: YopE ₁ . ₁₃₈ -domains of mouse Card Rig1. Titration of bacteria added to the cells (III: specified in the MH) was performed for each strain, and IFN stimulation was assessed by adding cell supernatant to an IFN reporter cell line based on the activity of secreted alkaline phosphatase (IV: OP650), which is under the control of the IISG54 promoter , consisting of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

Fig. 22 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - STING pathway. Delivery of enzymes that form cyclic dinucleotides leads to the induction of type I IFN in the IFN-reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE1_ ₁₃₈ - P. aeruginosa WspR (with an adapted stem domain). Titration of the bacteria added to the cells (III: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (IV: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54, enhanced by a multimeric ISRE.

Fig. 23 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - STING pathway. Delivery of cyclic dinucleotide-forming enzymes leads to the induction of type I IFN in the IFN-reporter cell line B16F1O. B16F10 reporter cells were left untreated (I) or infected with II: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on a pBadMycHisA-derived plasmid III: YopE1_ ₁₃₈ - V. cholerae DncV, IV: YopE1_ ₁₃₈ - B. cereus DisA-like protein, V : YopE1_ ₁₃₈ -cGAS anemone or VI: YopE1_ ₁₃₈ - MycHis. Titration of the bacteria added to the cells (VII: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VIII: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 24 - T388-dependent secretion of IRF3 into the culture supernatant. In vitro secretion experiment I: Y. enterocolitica AHOPEMAT+YopE1. ₁₃₈ - BH3 tBID mouse and II: Y. enterocolitica AHOPEMAT+YopE1. ₁₃₈ Mouse IRF3 Ser397Asp. The protein content of total bacterial lysates (A) and precipitated culture supernatants (B) was analyzed by Western blotting using an anti-YopE antibody. The numbers presented indicate the molecular weight in kDa at the corresponding height.

Fig. 25 - delivery of proteins that induce a type I interferon response using the bacterial T3SS into immune cells - the Rig1 and STING pathway. Delivery of mouse Card Rig1 domains and cyclic dinucleotide-forming enzymes results in the induction of type I IFN in the IFN reporter cell line RAW264.7. RAW264.7 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on a pBadMycHisA-derived plasmid II: YopE1_ ₁₃₈ - V. cholerae DncV, III: YopE1_ ₁₃₈ - B. cereus DisA-like protein, IV: YopE1 -138- anemone cGAS or V: YopE1_ ₁₃₈ - mouse Card Rigl domains. Titration of the bacteria added to the cells (VI: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VII: OP650), which is under the control of the IISG54 promoter, consisting of the IFN-inducible ISG54 promoter, enhanced by a multimeric ISRE.

Fig. 26 - colonization of the tumor with injected i.v. strains of Y. enterocolitica in the B16F10 breast cancer allograft model. Wild-type C57BL/6 mice with s.c. allograft of melanoma cancer cells B16F10 i.v. injected I: PBS, II: 1x10 ⁷ Y. enterocolitica dHOPEMT, III: Y. enterocolitica dHOPEMT+pYV-YopE1_ ₁₃₈ - CARD1.246 RIG1 mice or IV: Y. enterocolitica dHOPEMT AHairpinI-VirF+pYV-YopE1. ₁₃₈ - CARD1.246 RIG1 mouse, when the tumor reached a size of 100-315 mm ³ . The number of bacteria in tumors is reported as colony-forming units (CFU) per g of tissue (V). Counting was performed on tumors on days 5 or 8 postinfection. Each dot represents an individual mouse. The horizontal dotted line represents the detection limit.

Fig. 27 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - RIG1. Delivery of human and mouse RIG1 CARD domains results in the induction of type I IFN in the B16F10 IFN reporter cell line. B16F10 cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₅ human RIG1, III: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₆ mouse RIG1, IV: YopE ₁ . ₁₃₈ -domains CARD1.229 mouse RIG1, V: YopE ₁ . ₁₃₈ ^-CA domains of mouse RD1-218 RIG1. Titration of bacteria added to the cells (VI: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VII: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

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Fig. 28 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - RIG1. Delivery of human and mouse RIG1 CARD domains results in the induction of type I IFN in the IFN reporter RAW cell line. RAW reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₅ human RIG1, III: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₆ mouse RIG1, IV: YopE ₁ . ₁₃₈ domains CARD1- ₂₂₉ RIG1 mouse, V: YopE _1-138 domains CARD _1-218 RIG1 mouse. Titration of bacteria added to the cells (VI: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VII: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 29 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - MDA5 pathway. Delivery of mouse MDA5 results in the induction of type I IFN in the IFN reporter cell line B16F1O. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ . ₁₃₈ -MDA5 ₁ . ₂₉₄ mice, III: YopE ₁ - ₁₃₈ - MDA51 - ₂₃₁ mice. Titration of bacteria added to the cells (IV: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (V: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 30 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - MAVS. Delivery of CARD MAVS results in the induction of type I IFN in the IFN reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₆ mouse RIG1, III: YopE1. ₁₃₈ -cGAS ₁₆₁ . ₅₂₂ people, IV: YopE1. ₁₃₈ -CARD1. ₁₀₀ MAVS person. Titration of bacteria added to the cells (V: indicated in the MS) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VI: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 31 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - MAVS. Delivery of CARD MAVS results in the induction of type I IFN in the IFN-reporter macrophage cell line RAW. RAW macrophage reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ . ₁₃₈ -domains CARD1_ ₂₄₆ mouse RIG1, III: YopE1. ₁₃₈ -CGAS ₁₆₁ . ₅₂₂ humans, IV: YopE1.138-CARD1.100 human MAVS. Titration of bacteria added to the cells (V: indicated in the MS) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VI: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter, enhanced by a multimeric ISRE.

Fig. 32 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - STING pathway. Delivery of cyclic dinucleotide-forming enzymes leads to the induction of type I IFN in the IFN-reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ - ₁₃₈ -cGAS anemone, III: YopE ₁ . ₁₃₈ -cGAS ₆₀ . ₄₂₂ anemones, IV: YopE ₁ . ₁₃₈ -cGAS ₁₆₁ .5 ₂₂ humans, V: YopE1-138-CdaAioi-273 Listeria, VI: YopE1-138- DncV V. cholerae, VII: YopE1-138-DisA-like protein B. cereus. Titration of bacteria added to cells (VIII: designated as M3) was performed for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (IX: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 33 - delivery of proteins that induce a type I interferon response using the bacterial T3SS - STING pathway. Delivery of cyclic dinucleotide-forming enzymes leads to the induction of type I IFN in the IFN-reporter macrophage cell line RAW. RAW macrophage reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE ₁ - ₁₃₈ -cGAS anemone, III: YopE ₁ . ₁₃₈ -cGAS ₆₀ . ₄₂₂ anemones, IV: YopE ₁ . ₁₃₈ -cGAS ₁₆₁ .5 ₂₂ people, V: YopE ₁ . ₁₃₈ -CdaA ₁₀₁ . ₂ 7 ₃ Listeria, VI: YopE ₁ - ₁₃₈ - DncV V. cholerae, VII: YopE ₁ . ₁₃₈ -DisA-like protein of B. cereus. Titration of bacteria added to cells (VIII: designated as M3) was performed for each strain, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (IX: OP650), which is under the control of the IISG54 promoter, consisting of the IFN-inducible ISG54 promoter. enhanced by a multimeric ISRE.

Fig. 34 - Delivery of type I interferon response-inducing proteins by bacterial T3SS and comparison with small molecule STING agonists. Delivery of enzymes that form cyclic dinucleotides leads to the induction of type I IFN in the IFN-reporter cell line B16F10. B16F10 reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on a pBadMycHisA-derived plasmid II: YopE ₁ - ₁₃₈ -cGAS anemone, III:

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Human YopE1.138-cGAS161.522, IV: YopE ₁ . ₁₃₈ -domains of mouse CARD1_ ₂₁₈ RIG1. Titration of bacteria added to the cells (V: indicated in the MH) was carried out, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VI: OP650), which is under the control of the IISG54 promoter, consisting of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

Fig. 35 - Delivery of type I interferon response-inducing proteins by bacterial T3SS and comparison with small molecule STING agonists. Delivery of cyclic dinucleotide leads to the induction of type I IFN in the IFN reporter cell line B16F10. B16F10 reporter cells were treated with the small molecule STING agonist 2'3'-c-di-AM(PS)2 (Rp,Rp). Titration of the compound (I: indicated in micromoles) was carried out, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (II: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

Fig. 36 - Delivery of type I interferon response-inducing proteins by bacterial T3SS and comparison with small molecule STING agonists. Delivery of cyclic dinucleotide-forming enzymes leads to the induction of type I IFN in the IFN reporter RAW cell line. RAW reporter cells were infected with I: Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid II: YopE1_ ₁₃₈ -cGAS anemone, III: YopE1_ ₁₃₈ cGAS ₁₆₁ - ₅₂₂ human, IV: YopE ₁ . ₁₃₈ CARD1 domains. ₂₁₈ RIG1 mouse). Titration of bacteria added to the cells (V: indicated in the MH) was carried out, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (VI: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter, enhanced by a multimeric ISRE.

Fig. 37 - Delivery of type I interferon response-inducing proteins by bacterial T3SS and comparison with small molecule STING agonists. Delivery of cyclic dinucleotide leads to the induction of type I IFN in the IFN reporter RAW cell line. RAW reporter cells were treated with the small molecule STING agonist 2'3'-c-di-AM(PS)2 (Rp,Rp). Titration of the compound (I: indicated in micromoles) was carried out, and IFN stimulation was assessed based on the activity of secreted alkaline phosphatase (II: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE.

Fig. 38 - Delivery of type I interferon response-inducing proteins by bacterial T3SS and evidence of dependence on T3SS-RIG1 and MAVS. Delivery of CARD RIG1 or CARD MAVS domains fused to _YopE1-138 results in the induction of type I IFN in the IFN-reporter cell line RAW, which is strictly T3SS-dependent. RAW reporter cells were infected with I: Y. enterocolitica AHOPEMAT, or II: Y. enterocolitica AHOPEMAT-yopB, or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid III: YopE ₁ . ₁₃₈ -domains of mouse CARD1_ ₂₄₆ RIG1, V: YopE1_ ₁₃₈ CARD1_ ₁₀₀ human MAVS, or Y. enterocolitica AHOPEMAT-yopB, encoded on a plasmid derived from pBadMycHisA IV: YopE _{1_ 138} -domains of mouse CARD1_ ₂₄₆ RIG1, VI: YopE1_ ₁₃₈ -C ARD1_ ₁₀₀ MAVS person. Titration of the bacteria added to the cells (VII: indicated in the MH) was carried out for each strain, and IFN stimulation was assessed based on the activity of secreted Lucia luciferase (VIII: OP650), which is under the control of the I-ISG54 promoter, consisting of the IFN-inducible promoter ISG54 enhanced by a multimeric ISRE.

Fig. 39 - delivery of type I interferon response-inducing proteins by bacterial T3SS in a crude mixture of cells from a tumor isolate - RIG1. Delivery of RIG1 CARD domains fused to _YopE1-138 results in the induction of type I IFN in a crude tumor isolate. Wild-type Balb/C mice with s.c. allograft EMT6 breast cancer cells were killed when the tumor reached a volume of >200 mm ³ . Tumors were ground, digested, and plated as a single cell suspension in 24-well plates. Such cells from two different tumors were infected with I and III: Y. enterocolitica _{AHOPEMAT or II and IV: Y. enterocolitica AHOPEMAT, encoded on the pBadMycHisA-derived plasmid YopE 1_138 -domains CARD1_246} RIG1 of the mouse. Titration of bacteria added to the cells (V: indicated in the MoH) was performed for each strain, and IFN stimulation was assessed using an interferon beta ELISA (VI: picogram/milliliter). Dashed lines represent the corresponding untreated tumors, I/II and III/IV each representing cells derived from the same tumor.

Fig. 40 - tumor progression in wild-type Balb/C mice with s.c. allograft of EMT6 breast cancer cells. Wild-type Balb/C mice with s.c. Allograft EMT6 breast cancer cells were injected intratumorally with PBS when the tumor reached a size of 60–130 mm ³ . The day of the first intratumoral injection of PBS was designated as day 0, and treatment was performed on d0, d1, d5, d6, d10, and d11. Tumor volume was measured over the following days (I: day −11 to day 80 after the first bacterial injection) using a caliper. Relative tumor volume (tumor volume on the corresponding day divided by tumor volume in d0) in mm ³ is shown as log-2 transformed data (II) for each mouse.

Fig. 41 - tumor progression in wild-type Balb/C mice with s.c. allograft of EMT6 breast cancer cells. Wild-type Balb/C mice with s.c. 9 044058 EMT6 breast cancer cell allograft was injected intratumorally with 7.5x10 ⁷ Y. enterocolitica dHOPEMT when the tumor reached a size of 60-130 mm ³ . The day of the first intratumoral bacterial injection was designated as day 0, and treatment was performed on d0, d1, d5, d6, d10, and d11. Tumor volume was measured over the following days (I: day −11 to day 80 after the first bacterial injection) using a caliper. Relative tumor volume (tumor volume on the corresponding day divided by tumor volume in d0) in mm ³ is shown as log-2 transformed data (II) for each mouse.

Fig. 42 - tumor progression in wild-type Balb/C mice with s.c. allograft of EMT6 breast cancer cells. Wild-type Balb/C mice with s.c. An allograft of EMT6 breast cancer cells was intratumorally injected with 7.5x10 ⁷ Y. enterocolitica dHOPEMT, encoded on the pBadMycHisA-derived YopE ₁ plasmid. ₁₃₈ CARD1 domains. ₂ 4 ₆ RIG1 mice when the tumor reached a size of 60-130 mm ³ . The day of the first intratumoral bacterial injection was designated day 0, and treatment was performed on d0, d1, d5, d6, d10, and d11. Tumor volume was measured over the following days (I: day −11 to day 80 after the first bacterial injection) using a caliper. Relative tumor volume (tumor volume on the corresponding day divided by tumor volume in d0) in mm ³ is shown as log-2 transformed data (II) for each mouse.

Fig. 43 - tumor progression in wild-type Balb/C mice with s.c. allograft of EMT6 breast cancer cells. Wild-type Balb/C mice with s.c. An allograft of EMT6 breast cancer cells was intratumorally injected with 7.5x10 ⁷ Y. enterocolitica dHOPEMT encoded on the human pBadMycHisA-derived plasmid YopE1.138-cGAS161.522 when the tumor reached a size of 60-130 mm ³ . The day of the first intratumoral bacterial injection was designated as day 0, and treatment was performed on d0, d1, d5, d6, d10, and d11. Tumor volume was measured over the following days (I: day −11 to day 80 after the first bacterial injection) using a caliper. Relative tumor volume (tumor volume on the corresponding day divided by tumor volume in d0) in mm ³ is shown as log-2 transformed data (II) for each mouse.

Fig. 44 - tumor progression in wild-type Balb/C mice with repeated antigen stimulation s.c. on the opposite side with EMT6 breast cancer cells after the first complete remission. Wild-type Balb/C mice with s.c. Allograft EMT6 breast cancer cells were treated as described above (FIGS. 40-43), intratumorally with 7.5x10 ⁷ II: Y. enterocolitica dHOPEMT encoded on the pBadMycHisA-derived plasmid YopE ₁ . ₁₃₈ -domains of mouse CARD1-246 RIG1, W: Y. enterocolitica dHOPEMT, encoded on the YopE ₁ plasmid derived from pBadMycHisA. ₁₃₈ -cGAS ₁₆₁ . ₅₂₂ people when the tumor reached a size of 60-130 mm ³ . The day of intratumoral bacterial injection was designated as day 0. Mice with complete tumor regression (or I: naïve mice as controls) were injected s.c. on the contralateral side. EMT6 breast cancer cell allograft. Tumor volume was measured over the following days (IV: up to day 80 after the first bacterial injection) using a caliper. The absolute tumor volume (V) in mm ³ for each mouse is indicated.

Fig. 45 - tumor progression in wild-type Balb/C mice with s.c. allograft of EMT6 breast cancer cells. Wild-type Balb/C mice with s.c. allograft of breast cancer cells EMT6 i.v. injected I: PBS or 5x10 ⁶ II: Y. enterocolitica dHOPEMT, III: Y. enterocolitica dHOPEMT pYV-YopE1-138-(BH3 tBID)2, IV: Y. enterocolitica dHOPEMT AHairpinI-VirF pYV-YopE1-1 ₃₈ (BH3 tBID )2, V: Y. enterocolitica dHOPEMT AHairpinI-VirF Aasd pYV-asd-YopE ₁ . ₁₃₈ -(BH3 tBID)2, when the tumor reached a size of 80-250 mm ³ . Day v.v. bacterial injections were designated as day 0, all mice received i.p. (intraperitoneal) Desferal in d-1. Tumor volume was measured over the following days (VI: day 0 to day 15 after the first bacterial injection) using a caliper. The median tumor volume (VII) in mm ³ is indicated.

Fig. 46 - tumor progression in wild-type C57BL/6 mice with s.c. allograft of B16F10 melanoma cells. Wild-type C57BL/6 mice with s.c. allograft B16F10 melanoma cells were injected intratumorally with I: PBS or 7.5x10 ⁷ II: Y. enterocolitica dHOPEMT, III: encoded on the pBadMycHisA-derived plasmid YopE ₁ . ₁₃₈ -domains CARD1-246 of mouse RIG1, IV: Y. enterocolitica dHOPEMT, encoded on the pBadMycHisA-derived plasmid YopE ₁ . ₁₃₈ -cGAS ₁₆₁ . ₅₂₂ people when the tumor reached a size of 60-130 mm ³ . The day of the first intratumoral bacterial injection was designated day 0, and treatment was performed on d0, d1, d2, d3, d6, and d9. Tumor volume was measured over the following days (V: days) using a caliper. The average tumor volume (VI) in mm ³ is indicated.

Fig. 47 - biodistribution of Y. enterocolitica subsp. palearctica in the B16F10 mouse melanoma allograft model: assessment of physical appearance. I: Days, II: proportion of mice with score, III: Y. enterocolitica MRS40 WT (wild type), IV: Y. enterocolitica AyopH,O,P,E,M,T. The arrow points to the day v.v. injections of 2x10 ⁵ bacteria.

Fig. 48 - biodistribution of Y. enterocolitica subsp. palearctica in the B16F10 mouse melanoma allograft model: behavioral assessment. I: Days, II: proportion of mice with score, III: Y. enterocolitica MRS40 WT, IV: Y. enterocolitica AyopH,O,P,E,M,T. The arrow points to the day v.v. infections 2x10 ⁵ bacteria.

Fig. 49 - biodistribution of Y. enterocolitica subsp. palearctica in the B16F10 mouse melanoma allograft model: body weight of mice. The body weight of mice was assessed daily after i.v. infections

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AyopH,O,P,E,M,T. The arrow points to the day v.v. infections 2x10 ⁵ bacteria.

Fig. 50 - biodistribution of Y. enterocolitica subspecies palearctica in the B16F10 mouse melanoma allograft model: biodistribution of Y. enterocolitica AyopH,O,P,E,M,T. Organ counts at the indicated times were assessed by organ homogenization, serial dilution, and enumeration of the resulting colony-forming units (CFU). Day v.v. bacterial injections were designated as day 0, all mice received i.p. Desferal in d-1. I: Y. enterocolitica AyopH,O,P,E,M,T, II: CFU per 1 g of tissue or ml of blood, III: day 1, IV: day 4, V: blood, VI: spleen, VII: liver, VIII: lungs, IX: tumor. * denotes a mouse that had no visible tumor.

Fig. 51 - biodistribution of Y. enterocolitica subspecies palearctica on the B16F10 mouse melanoma allograft model: biodistribution of Y. enterocolitica MRS40 WT. Organ counts at the indicated times were assessed by organ homogenization, serial dilution, and enumeration of the resulting colony-forming units (CFU). Day v.v. bacterial injections were designated as day 0, all mice received i.p. Desferal in d-1.1: Y. enterocolitica MRS40 WT, II: CFU per 1 g of tissue or ml of blood, III: day 1, IV: day 4, V: blood, VI: spleen, VII: liver, VIII: lungs, IX : tumor.

Fig. 52 - delivery of type I interferon response-inducing proteins by bacterial T3SS - MAVS delivered by bacterial T3SS functions independently of endogenous MAVS.

Delivery of CARD MAVS by T3SS results in the induction of type I IFN in the IFN-reporter (luciferase) macrophage cell line RAW MAVS ^KO . RAW MAVS ^KO macrophage reporter cells were infected with I: Y. enterocolitica AHOPEMAT or II: Y. enterocolitica AHOPEMAT-yopB, III: Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid YopE _1-138 -CARD _1-100 human MAVS or IV: Y. enterocolitica AHOPEMAT-yopB, encoded on the pBadMycHisA-derived plasmid YopE _1-138 -CARD _1-100 human MAVS. Titration of bacteria added to cells (V: indicated in the MS) was carried out for each strain, and IFN stimulation was assessed based on luciferase activity (VI: REL - relative luminescence units), which is under the control of the I-ISG54 promoter, consisting of IFN- inducible ISG54 promoter enhanced by a multimeric ISRE.

Detailed Description of the Invention

The present invention relates to recombinant virulence-attenuated Gram-negative bacterial strains and their use in a method of treating cancer, such as a malignant solid tumor, in a subject.

For the purpose of interpreting this specification, the following definitions will apply and, where applicable, terms used in the singular will also include the plural and vice versa. It should be understood that the terminology used herein is intended solely to describe specific implementations and is not intended to be limiting.

The term gram-negative bacterial strain as used herein includes the following bacteria:

Aeromonas salmonicida, Aeromonas hydrophila, Aeromonas veronii, Anaeromyxobacter dehalogenans, Bordetella bronchiseptica, Bordetella parapertussis, Bordetella pertussis, Bradyrhizobium japonicum, Burkholderia cenocepacia, Burkholderia cepacia, Burkholderia mallei, Burkholderia pseudomallei, Chlamydia muridarum, Chlamydia trachmo atis, Chlamydophila abortus, Chlamydophila pneumoniae, Chromobacterium violaceum , Citrobacter rodentium, Desulfovibrio vulgaris, Edwardsiella tarda, Endozoicomonas elysicola, Erwinia amylovora, Escherichia albertii, Escherichia coli, Lawsonia intracellularis, Mesorhizobium loti, Myxococcus xanthus, Pantoea agglomerans, Photobacterium damselae, Photorhabdus luminescens, Photorabdus temper ate, Pseudoalteromonas spongiae, Pseudomonas aeruginosa, Pseudomonas plecoglossicida, Pseudomonas syringae, Ralstonia solanacearum, Rhizobium sp, Salmonella enterica and other Salmonella sp. Shigella flexneri and others Shigella sp, Sodalis glossinidius, Vibrio alginolyticus, Vibrio azureus, Vibrio campellii, Vibrio caribbenthicus, Vibrio harvey, Vibrio parahaemolyticus, Vibrio tasmaniensis, Vibrio tubiashii, Xanthomonas axonopodis, Xanthomonas campestris, Xanthomonas oryzae , Yersinia enterocolitica, Yersinia pestis, Yersinia pseudotuberculosis.

Preferred gram-negative bacterial strains according to the present invention are gram-negative bacterial strains that belong to the families Enterobacteriaceae and

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Pseudomonadaceae. The gram-negative bacterial strain of the present invention is typically used to deliver heterologous proteins via a bacterial T3SS into eukaryotic cells in vitro and/or in vivo, preferably in vivo.

The term recombinant virulence-attenuated gram-negative bacterial strain as used herein means a recombinant virulence-attenuated gram-negative bacterial strain that has been genetically transformed with a nucleotide molecule, such as a vector. The virulence of such a recombinant gram-negative bacterial strain is usually attenuated as a result of deletion of bacterial effector proteins having virulence activity, which are transported by one or more bacterial proteins that are part of the secretion system apparatus. Such effector proteins are delivered by the secretion system apparatus to host cells, in which they exhibit their virulence activity against various host proteins and cell mechanisms. Many different effector proteins are known that are transported by different types of secretion systems and exhibit a wide repertoire of biochemical activities that modulate the functions of host regulatory molecules. The virulence of the recombinant gram-negative bacterial strain herein may be further attenuated due to the absence of a siderophore normally or occasionally produced by the gram-negative bacterial strain, such that the strain does not produce a siderophore, eg, is deficient in siderophore production. Thus, in a preferred embodiment, a recombinant virulence-attenuated Gram-negative bacterial strain is used that lacks the siderophore normally or occasionally produced by the Gram-negative bacterial strain, such that the strain does not produce a siderophore, for example, is deficient in siderophore production, more preferably, is used Yersinia strain, in particular Y. enterocolitica MRS40 AyopH,O,P,E,M,T, Y. enterocolitica MRS40 AyopH,O,P,E,M,T AHairpinI-virF or Y. enterocolitica MRS40 AyopH,O,P ,E,M,T Aasd pYV-asd, with the absence of a siderophore, normally or sometimes produced by a gram-negative strain of bacteria, as a result of which the strain does not produce a siderophore, for example, it is deficient in the production of siderophore, in particular, it is deficient in the production of yersiniabactin. Y. enterocolitica MRS40 AyopH,O,P,E,M,T, which is deficient in the production of yersiniabactin, was described in publication WO02077249 and was deposited on September 24, 2001 under the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the Belgian Coordinated Collections of Microorganisms (BCCM); this strain was assigned the registration number LMG P-21013. The recombinant virulence-attenuated Gram-negative bacterial strain preferably does not produce at least one, preferably at least two siderophores, for example, is deficient in the production of at least one, preferably at least two siderophores, more preferably, the recombinant attenuated virulence, the Gram-negative bacterial strain does not produce a single siderophore.

The terms siderophore, iron siderophore, or iron chelator, as used interchangeably herein, refer to compounds with high affinity for iron, eg, small compounds with high affinity for iron.

Siderophores of gram-negative bacteria are, for example, enterobactin and dihydroxybenzoylserine, synthesized by Salmonella, Escherichia, Klebsiella, Shigella, Serratia (but used by all enterobacteria), pyoverdines, synthesized by Pseudomonas, vibriobactin, synthesized by Vibrio, acinetobactin and acinetoferrin, synthesized by Acinetobacter, yersiniabactin and aerobactin, synthesized by Yersinia, ornibactin synthesized by Burkholderia, salmochelin synthesized by Salmonella, aerobactin synthesized by Escherichia, Shigella, Salmonella and Yersinia, alkaligin synthesized by Bordetella, bizukaberin synthesized by Vibrio.

Siderophores include hydroxamate, catecholate, and mixed-ligand siderophores. To date, several siderophores have been approved for use in humans, namely 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, Defridget, Desifer), and deferiprone (Ferryprox).

The term endogenous protein essential for growth as used herein refers to proteins from a recombinant virulence-attenuated Gram-negative bacterial strain without which the Gram-negative bacterial strain cannot grow. Endogenous proteins required for growth are, for example, an enzyme required for amino acid production, an enzyme involved in peptidoglycan biosynthesis, an enzyme involved in LPS (lipopolysaccharide) biosynthesis, an enzyme involved in nucleotide synthesis, or a translation initiation factor.

The term amino acid production enzyme as used herein refers to enzymes that are associated with amino acid production of a recombinant virulence-attenuated Gram-negative bacterial strain and without which the Gram-negative bacterial strain cannot grow. The enzymes required for amino acid production are e.g.

- 12 044058 aspartate beta-semialdehyde dehydrogenase (asd), glutamine synthetase (glnA), tryptophanyl-tRNA synthetase (trpS) or serine hydroxymethyl transferase (glyA), or transketolase 1 (tktA), transketolase 2 (tktB), ribulose phosphate 3 -epimerase (rpe), ribose-5-phosphate isomerase A (rpiA), transaldolase A (talA), transaldolase B (talB), phosphoribosyl pyrophosphate synthase (prs), ATP phosphoribosyltransferase (hisG), bifunctional histidine biosynthetic protein HisIE (hisI) . -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 (agoA), chorismate synthase (agoC), P protein (pheA), T protein (tyrA), aromatic amino acid aminotransferase (tyrB), phospho-2-dehydro-3-deoxyheptonataldolase (aroG), phospho-2 -dehydro-3-deoxyheptonaldolase (aroH), phospho-2-dehydro-3-deoxyheptonaldolase (aroF), quinate/shikimate dehydrogenase (ydiB), ATP-dependent isoenzyme 6-phosphofructokinase 1 (pfkA), ATP-dependent isoenzyme 6- phosphofructokinase 2 (pfkB), fructose bisphosphate aldolase class 2 (fbaA), fructose bisphosphate 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-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 I (sdaA), L-serine dehydratase 2 (sdaB), L-threonine dehydratase catabolic (tdcB), L-threonine dehydratase biosynthetic (ilvA), L-serine dehydratase (tdcG) 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), biosynthetic L-threonine dehydratase (ilvA), large subunit acetolactate synthase isoenzyme 3 (ilvI), acetolactate synthase isoenzyme 3 small subunit (ilvH), acetolactate synthase isoenzyme 1 small subunit (ilvN), acetolactate synthase isoenzyme 2 small subunit (ilvM), ketol acid reductoisomerase (NADP(+)) (ilvC), dehydrate zu dihydroxy acids (ilvD), branched-chain amino acid aminotransferase (ilvE), bifunctional aspartokinase/homoserine dehydrogenase 1 (thrA), bifunctional aspartokinase/homoserine dehydrogenase 2 (metL), 2-isopropylmalate synthase (leuA), glutamate-pyruvate aminotransferase (alaA), aspartate -aminotransferase (aspC), bifunctional aspartokinase/homoserine dehydrogenase 1 (thrA), bifunctional aspartokinase/homoserine dehydrogenase 2 (metL), lysine-sensitive aspartokinase 3 (lysC), aspartate-semialdehyde dehydrogenase (asd), 2-keto-3-deoxy- galactonate aldolase (yagE), 4-hydroxy-tetrahydrodipicolinate synthase (dapA), 4-hydroxy-tetrahydrodipicolinate reductase (dapB), 2,3,4,5-tetrahydropyridine-2,6dicarboxylate N-succinyltransferase (dapD), succinyl-diaminopimelate -desuccinylase (dapE), diaminopimelate epimerase (dapF), putative lyase (yjhH), acetylornithine/succinyldiaminopimelate aminotransferase (argD), citrate synthase (gltA), aconitate hydratase B (acnB), aconitate hydratase A (acnA), ^characterized putative aconitate hydratase ( ybhJ), isocitrate dehydrogenase (icd), aspartate aminotransferase (aspC), glutamate-pyruvate aminotransferase (alaA), large chain glutamate synthase [NADPH] (gltB), small chain glutamate synthase [NADPH] (gltD), glutamine synthetase (glnA), acetyltransferase Amino acid (Arga), acetylglutamatkinase (Argb), n-acetyl-gamma-glutumilphosphate reductase (Argc), acetylornitin/succinyl-pimino-aminotransferaza (Argd), acetylornitin-deacetylosis, and a circuit F Ornitinkarb MILTRANSFERAZ (Argf), chain I ornitinkarbamoilTransferase ( argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), glutamate 5-kinase (proB), gamma-glutamylphosphate reductase (proA), pyrroline-5-carboxylate reductase (proC), ornithine cyclodeaminase, leucine-tRNA ligase (leuS), glutamine-tRNA ligase (glnS), serine-tRNA ligase (serS), glycine-tRNA ligase beta subunit (glyS), glycine-tRNA ligase alpha subunit (glyQ), tyrosine-tRNA ligase (tyrS), threonine-tRNA ligase (thrS) , phenylalanine-tRNA ligase alpha subunit (pheS), phenylalanine-tRNA ligase beta subunit (pheT), arginine-tRNA ligase (argS), histidine-tRNA ligase (hisS), valine-tRNA ligase (valS), alanine-tRNA ligase (alaS), isoleucine-tRNA ligase (ileS), proline-tRNA ligase (proS), cysteine-tRNA ligase (cysS), asparagine-tRNA ligase (asnS), aspartate-tRNA ligase (aspS), glutamate-tRNA ligase ( gltX), tryptophan-tRNA ligase (trpS), glycine-tRNA ligase beta subunit (glyS), methionine-tRNA ligase (metG), lysine-tRNA ligase (lysS). The preferred enzymes required for amino acid production are

- 13 044058 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, more preferred enzymes 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, most preferred the enzyme is asd.

The terms gram-negative bacterial strain deficient in the production of an amino acid required for growth and auxotrophic mutant are used interchangeably herein to refer to gram-negative bacterial strains that cannot grow in the absence of at least one exogenously provided essential amino acid or its precursor. An amino acid for which the strain is deficient in production is, for example, aspartate, meso2,6-diaminopimelic acid, aromatic amino acids or leucine-arginine. Such a strain can be obtained, for example, as a result of deletion of the aspartate beta-semialdehyde dehydrogenase (Δasd) gene. This auxotrophic mutant cannot grow in the absence of exogenous meso-2,6diaminopimelic acid. A mutation, for example, deletion of the aspartate-beta-semialdehyde dehydrogenase gene, is herein preferred for a Gram-negative bacterial strain deficient in the production of an amino acid required for growth according to the present invention.

The term gram-negative bacterial strain deficient in the production of adhesion proteins that bind to the eukaryotic cell surface or extracellular matrix refers to mutant gram-negative bacterial strains that do not express at least one adhesion protein compared to the adhesion proteins expressed by the corresponding wild-type strain. Adhesion proteins may include, for example, extended polymeric adhesion molecules such as pili/fimbriae, or non-fimbrial adhesins. Fimbrial adhesins include type 1 pili (such as E. coli Fim pili with the adhesin FimH), P pili (such as Pap pili with the PapG adhesin from E. colt), type 4 pili (such as the pilin protein, e.g. P. aeruginosa) or carlin (Csg proteins with the CsgA adhesin from S. enterica). Nonfimbrial adhesins include trimeric autotransporter adhesins such as YadA from Y. enterocolitica, BpaA (B. pseudomallei), Hia (H. influenzae), BadA (B. henselae), NadA (N. meningitidis), or UspA1 (M. catarrhalis) , as well as other autotransporter adhesins such as AIDA-1 (E. coli), as well as other adhesins/invasives such as InvA from Y. enterocolitica or intimin (E. coli), or members of the Dr family or the Afa family (E .coli). The terms YadA and InvA as used herein refer to proteins from Y. enterocolitica. The autotransporter YadA ⁷ binds to various forms of collagen as well as fibronectin, while the invasin InvA ⁸ binds to β-integrins in the eukaryotic cell membrane. If the gram-negative bacterial strain is a Y. enterocolitica strain, the strain is preferably deficient in InvA and/or YadA.

As used herein, the term family Enterobacteriaceae includes a family of gram-negative, rod-shaped, facultative anaerobic bacteria found in soil, water, plants and animals that are often present as pathogens in vertebrates. Bacteria of this family are characterized by similar physiology and demonstrate conservation of functional elements and genes of the corresponding genomes. Also, being oxidase negative, all members of this family ferment glucose, and most are nitrate reducing. The Enterobacteriaceae bacteria of the present invention can be any bacterium from this family and specifically includes, without limitation, bacteria from the following genera:

Escherichia, Shigella,

Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Serratia, Proteus, Erwinia, Morganella, Providencia or Yersinia.

- 14 044058

In more specific embodiments, the bacterium is of the species

Escherichia coli, Escherichia blattae,

Escherichia fergusonii, Escherichia hermanii, Escherichia vuneris, Salmonella enterica, Salmonella bongori, Shigella dysenteriae, Shigella flexneri, Shigella boydii, Shigella sonnei, Enterobacter aerogenes, Enterobacter gergoviae, Enterobacter sakazakii, Enterobacter cloacae, Enterobacter agglomerans, Klebsiella pneumoniae, Klebs iella oxytoca, Serratia marcescens , Yersinia pseudotuberculosis, Yersinia pestis, Yersinia enterocolitica, Erwinia amylovora, Proteus mirabilis, Proteus vulgaris, Proteuspenneri, Proteus hauseri, Providencia alcalifaciens or Morganella morganii.

Preferably, the gram-negative bacterial strain is selected from the group consisting of the genera

Yersinia, Escherichia, Salmonella, Shigella, 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 genera

Yersinia, Escherichia, Salmonella and Pseudomonas, most preferably from the group consisting of the genera Y^sinia and Salmonella, in particular Yersinia.

The term Yersinia as used herein includes all species of Yersinia, including Yersinia enterocolitica, Yersinia pseudotuberculosis and Yersinia pestis. Yersinia enterocolitica is preferred.

The term Salmonella as used herein includes all Salmonella species, including Salmonella enterica and S. bongori. Salmonella enterica is preferred.

A promoter as used herein refers to a nucleic acid sequence that regulates the expression of a transcription unit.

The promoter region is a regulatory region capable of binding to RNA polymerase in the cell and initiating transcription of the downstream (3' direction) coding sequence. The promoter region contains the transcription initiation site (usually determined by S1 nuclease mapping) as well as protein-binding domains (consensus sequences) responsible for RNA polymerase binding, such as the putative -35 region and the Pribnow box. The term operably linked, when describing the interaction between two nucleotides, such as sections of DNA, means nothing more than that the sections are operably linked to each other and located on the same nucleic acid fragment. A promoter is functionally associated with a structural gene if it controls transcription of the gene and is located on the same nucleic acid fragment as the gene. Typically the promoter is functional in the specified gram-negative bacterial strain, i.e. the promoter is capable of expressing the fusion protein of the present invention, i.e. the promoter is capable of expressing the fusion protein of the present invention without further use of genetic engineering or expression of additional proteins. Moreover, a functional promoter in nature should not be counter-regulated by the bacterial T3SS.

The term extrachromosomal genetic element as used herein means a genetic element, other than a chromosome, contained endogenously in a Gram-negative bacterial strain of the present invention, such as a virulence plasmid, or which is an exogenous genetic element with which a Gram-negative bacterial strain is transformed and which is transiently or stably integrated into chromosome or into a genetic element other than a chromosome contained endogenously, such as a virulence plasmid. Such extrachromosomal genetic element may be a vector, such as an expression vector, a vector for homologous recombination or other type of integration into a chromosome, or into a genetic element other than a chromosome contained endogenously, such as a virulence plasmid, DNA fragments for homologous recombination or other type of integration into a chromosome or into a non-chromosomal genetic element contained endogenously, such as a virulence plasmid or RNA element that directs a site-specific insertion into a chromosome or into a non-chromosomal genetic element contained endogenously, such as a virulence plasmid, such as CRISPR/ Cas9, and associated guide RNA.

The term temperature-sensitive RNA region as used herein refers to a temperature-sensitive non-coding RNA sequence that regulates the expression of associated genes. Typically, temperature-sensitive regions of RNA function through the formation of a secondary structure, such as an RNA hairpin loop, which forms stably at the repressive temperature and is unstable at the permissive temperature and which masks the RNA sequence,

- 15 044058 necessary for translation, such as a ribosome binding site, thereby regulating the expression of a gene associated with such RNA sequence necessary for translation.

The term RNA hairpin structure or portions thereof as used herein refers to the secondary structure of RNA formed by intramolecular base pairing that results in the formation of a stem-loop structure. Intramolecular base pairing, usually within a single strand of RNA, occurs due to complementary nucleotide sequences or parts thereof.

The term AraC-type DNA binding protein, also referred to as the AraC/XylS family, as used herein refers to bacterial transcriptional regulatory proteins that bind DNA through a helix-loop-helix motif. Most members of the AraC-type DNA-binding proteins are positive regulators of transcription and can be characterized by a minimal DNA-binding domain spanning a fragment of more than 100 residues containing two helix-loop-helix subdomains. AraC-type DNA binding proteins specifically include, but are not limited to: VirF, LcrF, YbtA, Rns, MxiE, AraC, XylS, ExsA, PerA, MmsR, RhaS, TcpN, HrpX, HrpB, GadX, HilC, HilD, MarA, CafR, FapR and InvF. Preferred are DNA-binding proteins of the AraC type involved in the regulation of virulence-related mechanisms, such as VirF, LcrF, YbtA, Rns, MxiE, ExsA, PerA, HrpX, HrpB, GadX, HilC, HilD, TcpN, CafR, FapR and InvF. More preferred are DNA-binding proteins of the AraC type involved in the regulation of the activity of the third type secretion system, such as VirF, LcrF, MxiE, ExsA, PerA, HrpX, HrpB, GadX, HilC, HilD and InvF, most preferred are VirF and/or LcrF.

The term delivery as used herein means the transport of a protein from a recombinant virulence-attenuated Gram-negative bacterial strain into a eukaryotic cell, including the steps of expression of the heterologous protein in the recombinant virulence-attenuated Gram-negative bacterial strain, secretion of the expressed protein or proteins from such recombinant virulence-attenuated Gram-negative bacterial strain, and translocation secreted protein or proteins by such a recombinant virulence-attenuated gram-negative strain of bacteria into the cytosol of a eukaryotic cell. Accordingly, the terms delivery signal or secretion signal, as used interchangeably herein, refer to a polypeptide sequence that can be recognized by the secretion and translocation system of a Gram-negative bacterial strain and directs the delivery of a protein from the Gram-negative bacterial strain into eukaryotic cells.

The term bacterial effector protein delivery signal as used herein refers to a bacterial effector protein delivery signal functional in a recombinant gram-negative bacterial strain, i.e. which causes secretion of a heterologous protein expressed in a recombinant gram-negative bacterial strain from such a recombinant gram-negative bacterial strain by a secretion system such as a type III, type IV or type VI secretion system, or translocation of a heterologous protein expressed in a recombinant gram-negative bacterial strain by such a recombinant gram-negative bacterial strain into the cytosol of a eukaryotic cell by a secretion system such as a type III, type IV, or type VI secretion system. The term bacterial effector protein delivery signal as used herein also includes a fragment of a bacterial effector protein delivery signal, i.e. shorter versions of the delivery signal, for example, a delivery signal containing up to 10, preferably up to 20, more preferably up to 50, even more preferably up to 100, in particular up to 140 amino acids of the delivery signal, for example, a naturally occurring delivery signal. Thus, a nucleotide sequence, such as, for example, a DNA sequence encoding a delivery signal from a bacterial effector protein, may encode a full-length delivery signal or a fragment thereof, the fragment typically containing up to 30, preferably up to 60, more preferably up to 150, more more preferably up to 300, in particular up to 420 nucleic acids.

As used herein, protein secretion refers to the transport of a heterologous protein outward across the cell membrane of a recombinant virulence-attenuated Gram-negative bacterial strain. Protein translocation refers to the transport of a heterologous protein from a recombinant virulence-attenuated Gram-negative bacterial strain across the plasma membrane of a eukaryotic cell into the cytosol of such a eukaryotic cell.

The term bacterial protein that is part of the secretion system apparatus as used herein refers to bacterial proteins that form important components of the bacterial type 3 secretion system (T3SS), type 4 secretion system (T4SS) and type 6 secretion system (T6SS), preferably the T3SS. Without such proteins, the corresponding secretion system is nonfunctional in translocating proteins into host cells, even if all other components of the secretion system and the bacterial effector protein to be translocated are still encoded and produced.

The term bacterial effector protein as used herein refers to bacterial proteins transported by secretion systems, eg, bacterial proteins that are part of the secretion system machinery, into host cells. Such effector proteins are delivered through the secretion system into the host cell, in which they exhibit, for example, virulence activity against various host proteins and cell apparatus. Many different effector proteins are known that are transported by different types of secretion systems and exhibit a wide repertoire of biochemical activities that modulate the functions of host regulatory molecules. The secretion systems include type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS). Some effector proteins (such as IpaC of Shigella flexneri) also belong to a class of bacterial proteins that are part of the secretion system machinery and mediate protein translocation. The recombinant virulence-attenuated Gram-negative bacterial strain herein typically contains bacterial proteins that form essential components of a bacterial type 3 secretion system (T3SS), a type 4 secretion system (T4SS), and/or a type 6 secretion system (T6SS), preferably a type 3 secretion system. type (T3SS). The term bacterial proteins that form essential components of a bacterial T3SS as used herein refers to proteins that naturally form an injectisome, such as an injection needle, or that are otherwise essential for its function in protein translocation into eukaryotic cells. Proteins that form the injectisome or are otherwise essential for its function during protein translocation 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 (external M-ring protein), SctJ, YscJ, MxiJ, PrgK, SsaJ, EscJ, HrcJ, HrcJ, FliF (internal M-ring protein), SctR, YscR, Spa24, SpaP, SpaP, SsaR, EscR, HrcR, HrcR, FliP (small export apparatus protein), SctS, YscS, Spa9 (SpaQ), SpaQ, SsaS, EscS, HrcS, HrcS, FliQ (small export apparatus protein), SctT, YscT, Spa29 (SpaR), SpaR, SsaT, EscT, HrcT, HrcT, FliR (small export apparatus protein), SctU , YscU, Spa40, SpaS, SpaS, SsaU, EscU, HrcU, HrcU, FlhB (switch protein of the export apparatus), SctV, YscV, MxiA, InvA, SsaV, EscV, HrcV, HrcV, FlhA (large protein of the export apparatus), SctK, YscK, MxiK, OrgA, HrpD (cytosolic accessory protein), SctQ, YscQ, Spa33, SpaO, SpaO, SsaQ, EscQ, HrcQA+B, HrcQ, FliM + FliN (C-ring protein), SctL, YscL, MxiN , OrgB, SsaK, EscL, Orf5, HrpE, HrpF, FliH (stator), SctN, YscN, Spa47, SpaL, InvC, SsaN, EscN, HrcN, HrcN, Flil (ATPase), SctO, YscO, Spal3, SpaM, Invl , SsaO, Orfl5, HrpO, HrpD, FliJ (Stalk), SctF, YscF, MxiH, PrgI, SsaG, EscF, HrpA, HrpY (needle filament protein), SctI, YscI, Mxil, PrgJ, Ssal, EscI, rOrf8, HrpB , HrpJ, (internal rod protein), SctP, YscP, Spa32, SpaN, InvJ, SsaP, EscP, Orfl6, HrpP, HpaP, FliK (needle length regulator), LcrV, IpaD, SipD (hydrophilic translocator, needle end protein), YopB, IpaB, SipB, SseC, EspD, HrpK, PopFl, 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, HraA (gatekeeper).

The term T6SS effector protein or bacterial T6SS effector protein as used herein refers to proteins that are naturally injected by T6S systems into the cytosol of eukaryotic cells or bacteria, and proteins that are naturally secreted by T6S systems that may, for example, form translocation pores in the eukaryotic membrane. The term T4SS effector protein or bacterial T4SS effector protein as used herein refers to proteins that are naturally injected by T4S systems into the cytosol of eukaryotic cells, and proteins that are naturally secreted by T4S systems that may, for example, form a translocation pore in the eukaryotic membrane. The term T3SS effector protein or bacterial T3SS effector protein as used herein refers to proteins that are naturally injected by T3S systems into the cytosol of eukaryotic cells, and proteins that are naturally secreted by T3S systems that may, for example, form a translocation pore in the eukaryotic membrane (including pore-forming translocators (such as Yersinia YopB and YopD) and needle tip proteins such as Yersinia LcrV). Preferably, proteins are used that are naturally injected by T3S systems into the cytosol of eukaryotic cells. These virulence factors paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. T3S effectors exhibit a wide repertoire of biochemical activities, modulate the function of critical host regulatory molecules, and turn on

- 17 044058

AvrA, AvrB, AvrBs2,

AvrBS3, AvrBsT, AvrD, AvrDl, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpml, AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA family proteins , HopAB2, HopAOl, Hopll, HopMl, HopNl, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, HopUl, HsvB, IcsB, IpaA, IpaB, IpaC, IpaH, IpaH7.8, IpaH9.8, IpgBl, IpgB2, IpgD, LcrV, Map, OspCl, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXol, PthXo6, PthXo7, SifA, SifB, SipA/SspA, SipB, SipC/SspC, SipD/SspD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SpiC/SsaB, SptP, SpvB, SpvC, SrfH, Srfl, Sse, SseB, SseC, SseD, SseF, SseG, SseESrfH, SseJ, SseKl, SseK2, SseK3, SseL, SspHl, SspH2,

SteA, SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopB, YopD

YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA.

The term recombinant virulence-attenuated Gram-negative bacterial strain accumulating in a malignant solid tumor or recombinant virulence-attenuating Gram-negative bacterial strain accumulating in a malignant solid tumor herein means a recombinant virulence-attenuated Gram-negative bacterial strain that replicates in a malignant solid tumor, thereby increasing the number of bacteria of a given recombinant virulence-attenuated gram-negative strain of bacteria inside a malignant solid tumor. Surprisingly, it was discovered that a recombinant virulence-attenuated Gram-negative bacterial strain, after administration to a subject, accumulates specifically in a malignant solid tumor, i.e. accumulates specifically in an organ in which a malignant tumor is present, and the number of bacteria of a recombinant virulence-attenuated gram-negative strain of bacteria in organs in which a malignant solid tumor is not present is low or undetectable.

In the case of extracellular bacteria such as Yersinia, bacteria preferentially accumulate in the intercellular space formed between tumor cells. Intracellularly growing bacteria such as Salmonella will preferentially occupy tumor cells and will reside within such cells, although extracellular accumulation may also occur. The number of bacteria of a recombinant virulence-attenuated gram-negative strain of bacteria accumulated inside a malignant solid tumor can be, for example, in the range from 10 ⁴ to 10 ⁹ bacteria per 1 g of tumor tissue.

The term cancer as used herein 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 through the circulatory and lymphatic systems. There are several main types of cancer. Carcinoma is cancer that occurs in the skin or tissues lining or covering internal organs. Sarcoma is a cancer that occurs in bone, joint, fat, muscle, blood vessels, or other connective or supporting tissue. Leukemia is a cancer that originates in blood-forming tissue such as bone marrow and causes large numbers of abnormal blood cells to be produced and released into the blood. Lymphoma and multiple myeloma are types of cancer that start in cells of the immune system. Central nervous system cancer types are types of cancer that originate in the tissues of the brain and spinal cord. The term cancer as used herein includes solid tumors, i.e. malignant solid tumors, such as, for example, sarcomas, carcinomas and lymphomas, and non-solid tumors, such as, for example, leukemias (types of blood cancer). Malignant solid tumors are preferred. The term malignant solid tumor or malignant solid tumor sign as used herein refers to an abnormal mass of tissue that does not typically contain cysts or fluid areas. Solid tumors can be benign (non-cancerous) or malignant (cancerous). Malignant solid tumors are treated with the methods of the present invention. The different types of malignant solid tumors are named by the type of cells that form them. Examples of malignant solid tumors are sarcomas, carcinomas and lymphomas. Leukemias (blood cancers) do not usually form malignant solid tumors (as defined by the National Cancer Institute of the US National Health Center). Malignant solid tumors include, but are not limited to, an abnormal mass of cells that may originate from a variety of tissue types, such as the liver, colon, colon, rectum, skin, breast, pancreas, cervix, uterine corpus, bladder, gallbladder bladder, kidneys, larynx, lip, oral cavity, esophagus, ovary, prostate gland,

- 18 044058 stomach, testicles, thyroid gland or lungs and thus includes malignant solid tumors of the liver, colon, colon and rectum, skin, breast, pancreas, cervix, uterine body, bladder, gallbladder, kidney, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testicles, thyroid or lungs. Preferred malignant solid tumors that can be treated by the methods of the present invention are malignant solid tumors that originate from the skin, breast, liver, pancreas, bladder, prostate and colon and thus include malignant solid tumors of the skin, breast, liver, pancreas, bladder, prostate and colon. Equally preferred malignant solid tumors that can be treated by the methods of the present invention are malignant solid tumors associated with liver cancer, such as hepatocellular carcinoma.

The term bacterial effector protein that is virulent against eukaryotic cells as used herein refers to bacterial effector proteins that are transported by secretion systems into host cells in which they exert their virulence activity against various host proteins and cellular machinery. Many different effector proteins are known that are transported by different types of secretion systems and exhibit a wide repertoire of biochemical activities that modulate the functions of host regulatory molecules. The secretion systems include type 3 secretion system (T3SS), type 4 secretion system (T4SS), and type 6 secretion system (T6SS). It is important to note that some effector proteins that are virulent against eukaryotic cells (such as IpaC of Shigella flexneri) also belong to a class of bacterial proteins that are part of the secretion system apparatus. If a bacterial effector protein that is virulent against eukaryotic cells is also essential for the functioning of the secretion apparatus, such a protein is excluded from this definition. T3SS effector proteins that are virulent against eukaryotic cells refer to proteins such as

YopE, YopH, YopJ, YopM, YopO, YopP, YopT K enterocolitica, or OspF, IpgD, IpgBl Shigella flexneri, or SopE, SopB, SptP Salmonella enterica, or ExoS, ExoT, ExoU, ExoY P. aeruginosa, or Tir, Map , EspF, EspG, EspH, EspZ E. coli.

T4SS effector proteins that are virulent against eukaryotic cells refer to proteins such as

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, CeglO, Ceg23, Ceg29 Legionella pneumophila, or Bep A, BepB, BepC, BepD, BepE, BepF BepG Bartonella henselae, or VirD2, VirE2, VirE3, VirF Agrobacterium tumefaciens, or CagA I. Pylori, or Bordetella pertussis pertussis toxin.

T6SS effector proteins that are virulent against eukaryotic cells are proteins such as the Vibrio cholerae VgrG proteins (such as VgrG1).

The term T3SS effector protein that is virulent on eukaryotic cells or bacterial T3SS effector protein that is virulent on eukaryotic cells as used herein refers to proteins that are naturally injected by T3S systems into the cytosol of eukaryotic cells and proteins that are naturally secreted by T3S systems, which can, for example, form a translocation pore in the eukaryotic membrane, which are virulence factors against eukaryotic cells, i.e. proteins paralyze or reprogram the eukaryotic cell to the benefit of the pathogen. Effectors display a wide repertoire of biochemical activities and modulate the function of critical host regulatory mechanisms such as, for example, phagocytosis and the actin cytoskeleton, inflammatory signaling, apoptosis, endocytosis or secretory signaling pathways ²⁹ and include

AvrA, AvrB, AvrBs2, AvrBS3, AvrBsT, AvrD, AvrDl,

AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, AvrRpml,

AvrRpt2, AvrXv3, CigR, EspF, EspG, EspH, EspZ, ExoS, ExoT, GogB, GtgA, GtgE, GALA family proteins, HopAB2, HopAOl, Hopll, HopMl, HopNl, HopPtoD2,

- 19 044058

HopPtoE, HopPtoF, HopPtoN, HopUl, HsvB, IcsB, IpaA, IpaH, IpaH7.8, IpaH9.8, IpgBl, IpgB2, IpgD, LcrV, Map, OspCl, OspE2, OspF, OspG, OspI, PipB, PipB2, PopB, PopP2, PthXol, PthXo6, PthXo7, SifA, SifB, SipA/SspA„ SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SpiC/SsaB, SptP, SpvB, SpvC, SrfH, Srfl, Sse, SseB, SseC, SseD, SseF, SseG, SseVSrfH, SseJ, SseKl, SseK2, SseK3, SseL, SspHl, SspH2, SteA, SteB, SteC, SteD, SteE, TccP2, Tir, VirA, VirPphA, VopF, XopD, YopE, YopH, YopJ, YopM, YopO, YopP, YopT, YpkA.

Yersinia T3SS effector genes that are virulent in the eukaryotic cell and can be deleted/mutated, for example from Y. enterocolitica, are YopE, YopH, YopM, YopO, YopP (also called YopJ) and YopT ¹⁰ . Corresponding effector genes that are virulent in a eukaryotic cell may be deleted/mutated from Shigella flexneri (e.g. OspF, IpgD, IpgB1), Salmonella enterica (e.g. SopE, SopB, SptP), P. aeruginosa (e.g. ExoS, ExoT, ExoU, ExoY) or E. coli (for example, 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 proteins from AF386526.1 GI:18462515; effectors S. enterica from NC_016810.1 GI:378697983 or FQ312003.1 GI:301156631, P. aeruginosa effectors from AE004091.2 GI:110227054 or CP000438.1 GI:115583796 and E. coli effector proteins from NC_01 1601.1 GI:215485161) .

For the purposes of the present invention, genes are designated in lowercase and italics to distinguish them from proteins. When genes (indicated in lowercase and italics) follow the name of a bacterial species (such as E. coli), they indicate a mutation of the corresponding gene in the corresponding bacterial species. For example, YopE refers to the effector protein encoded by the yopE gene. Y. enterocolitica yopE is a Y. enterocolitica containing a mutation in the yopE gene.

As used herein, the terms polypeptide, peptide, protein, polypeptide and peptide are used interchangeably and refer to a series of amino acid residues linked to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. Preferred are proteins that contain an amino acid sequence containing at least 10 amino acids, more preferably at least 20 amino acids.

According to the present invention, a heterologous protein includes naturally occurring proteins or a portion thereof, and also includes artificially engineered proteins or a portion thereof. As used herein, the term heterologous protein refers to a protein or portion thereof other than a T3SS effector protein or an N-terminal fragment thereof to which the effector protein may be fused. In particular, a heterologous protein as used herein refers to a protein or part thereof that is not part of the proteome, i.e. the entire natural protein that makes up a specific recombinant virulence-attenuated gram-negative bacterial strain proposed and used in the present invention, for example, which does not belong to the proteome, i.e. the entire natural protein that makes up a specific strain of bacteria of the genus Yersinia, Escherichia, Salmonella or Pseudomonas. Typically the heterologous protein is derived from an animal, including a human. Preferably, the heterologous protein is a human protein or part thereof. More preferably, the heterologous protein is selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or regulation of apoptosis, cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, reporter proteins, factors transcription, proteases, small GTPases, GPCR (G-Protein Coupled Receptor)-related proteins, nanobody-nanobody fusions, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins. A particularly preferred heterologous protein is selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or regulation of apoptosis, cell cycle regulators, ankyrin repeat proteins, reporter proteins, small GTPases, proteins associated with with GPCRs, nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins. Even more preferred are heterologous proteins selected from the group consisting of proteins involved in the induction or regulation of interferon (IFN) response, proteins involved in apoptosis or regulation of apoptosis, cell cycle regulators and ankyrin repeat proteins. Most preferred are proteins involved in apoptosis or the regulation of apoptosis, or proteins involved in the induction or regulation of the interferon (IFN) response, in particular proteins involved in the induction or regulation of the interferon (IFN) response, such as heterologous animal proteins, preferably human proteins involved in apoptosis or regulation of apoptosis, or human proteins involved in the induction or regulation of interferon (IFN) response. Proteins involved in the induction or regulation of the interferon (IFN) response are preferably proteins involved in the induction or regulation of the type I interferon (IFN) response, more preferably human proteins involved in the induction or regulation of the type I interferon (IFN) response .

- 20 044058

In some embodiments, a gram-negative bacterial strain of the present invention contains two nucleotide sequences encoding identical or two different heterologous proteins fused independently in frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein.

In some embodiments, a Gram-negative bacterial strain of the present invention contains three nucleotide sequences encoding identical or three different heterologous proteins fused independently in frame to the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein.

The heterologous protein expressed by a recombinant virulence-attenuated Gram-negative bacterial strain typically has a molecular weight of 1 to 150 kDa, preferably 1 to 120 kDa, more preferably 1 to 100 kDa, most preferably 10 to 80 kDa.

In some embodiments, the heterologous protein portion comprises a heterologous protein domain. Thus, in some embodiments, the gram-negative bacterial strain of the present invention contains a nucleotide sequence encoding a heterologous protein domain. Preferably, the gram-negative bacterial strain of the present invention contains a nucleotide sequence encoding one or two heterologous protein domains, more preferably two heterologous protein domains.

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

The term heterologous proteins, which belong to the same functional class of proteins, as used herein refers to heterologous proteins that perform the same function, for example, heterologous proteins that have enzymatic activity, heterologous proteins that act in the same signaling pathway, such as, for example, regulation of the cell cycle, or are characterized by a common specific property, such as, for example, belong to the same class of bacterial effector proteins. Functional classes of proteins are, for example, proteins involved in apoptosis or regulation of apoptosis, proteins that act as cell cycle regulators, ankyrin repeat proteins, cell signaling proteins, proteins involved in the induction or regulation of interferon (IFN) response, reporter proteins, transcription factors, proteases, small GTPases, GPCR-associated proteins, nanobody-nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors, or viral proteins that act cooperatively in the biological process of establishing virulence against eukaryotic cells.

According to the present invention, the heterologous protein domain includes domains of naturally occurring proteins and also includes domains of artificially engineered proteins. As used herein, the term heterologous protein domain refers to a domain of a heterologous protein other than that of a T3SS effector protein, or a domain other than the domain containing its N-terminal fragment to which the effector protein can be fused to form a fusion protein. In particular, a heterologous protein domain herein refers to a heterologous protein domain that is not part of the proteome, i.e. the entire natural protein constituting a particular recombinant gram-negative bacterial strain proposed and used in the present invention, for example, which does not belong to the proteome, i.e. the entire natural protein that makes up a specific strain of bacteria of the genus Yersinia, Escherichia, Salmonella or Pseudomonas. Typically, the heterologous protein domain is derived from an animal, including a human. Preferably, the heterologous protein domain is a human protein domain. More preferably, the heterologous protein domain is a protein domain selected from the group consisting of proteins involved in apoptosis or regulation of apoptosis, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators, ankyrin repeat proteins, signal transduction proteins cells, reporter proteins, transcription factors, proteases, small GTPases, GPCR-related proteins, nanobody-nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins. A particularly preferred heterologous protein domain is a protein domain selected from the group consisting of proteins involved in apoptosis or the regulation of apoptosis, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators, ankyrin repeat proteins, reporter proteins, small GTPases, GPCR-associated proteins, nanobody fusion constructs, bacterial T3SS effectors, bacterial T4SS effectors, and viral proteins. In particular, even more preferred are heterologous protein domains selected from the group consisting of proteins involved in apoptosis or regulation of apoptosis, proteins involved in the induction or regulation of interferon (IFN) response, cell cycle regulators and ankyrin repeat proteins. The most preferred domains are those of proteins involved in the induction or regulation of

- 21 044058 interferon (IFN) response proteins, such as animal proteins involved in the induction or regulation of the interferon (IFN) response, preferably domains of heterologous human proteins involved in the induction or regulation of the interferon (IFN) response.

The term heterologous protein repeat domains as used herein means a fusion protein consisting of multiple repeats of a heterologous protein domain, which domains can either be directly fused to each other or a variable linker can be introduced between the domains, for example, a linker of size 1 to 30, preferably from 2 to 15, more preferably from 3 to 10 amino acids. Preferably, repeat identical domains or repeat domains are used which have an amino acid sequence identity of greater than 80%, typically greater than 85%, preferably greater than 90%, even more preferably greater than 95%, in particular greater than 96%, more particularly greater than 97 %, even more specifically, more than 98%, most specifically, more than 99%. Also preferred are identical domains that have 100% amino acid identity. Preferably, the fusion protein as defined herein contains two repeat domains, more preferably two identical repeat domains or two repeat domains that have greater than 90% amino acid sequence identity, preferably greater than 95%, most preferably 100%. The present invention also provides for more than two, for example three, four, five or six repeat domains.

The term two or more different heterologous protein domains as used herein means a fusion protein consisting of one or more repeats of at least two different heterologous protein domains, e.g., at least two heterologous protein domains that share 80% or less amino acid sequence identity, preferably 60% or less, more preferably 40% or less, and these domains can either be directly fused to each other or a variable linker can be introduced between the domains, for example a linker of size 1 to 30, preferably 2 to 15, more preferably 3 to 10 amino acids. Preferably, two domains of different heterologous proteins are contained in the fusion protein, as defined herein. The present invention also provides more than two, for example three, four, five or six domains of different heterologous proteins.

The heterologous protein domain expressed by a recombinant Gram-negative bacterial strain typically has a molecular weight of 1 to 50 kDa, preferably 1 to 30 kDa, more preferably 1 to 20 kDa, most preferably 1 to 10 kDa.

According to the present invention, proteins involved in the induction or regulation of the IFN response include, but are not limited to, cGAS, STING, TRIF, TBK1,

PSKepsilon, IRF3, TREX1, ¥PS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11,

DNA-PK, RIG1, MDA5, LGP2, 1Р8-1/МА¥8/cardif/¥18А, Trim25, Trim32, Trim56, riplet, TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR , TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSml4A, LRRFIP1, DHX9, DHX36, DHX29, DHX15, Ku70, IFNAR1, IFNAR2, TYK2, JAKI, ISGF3, IL10R2, IFNLR1, IFNGR1, IF NGR2, JAK2 , STAT4, cyclic dinucleotide-forming enzymes (cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP cyclases), such as WspR, DncV, DisA and DisA-like protein, CdaA, CdaS and cGAS or fragments thereof.

According to the present invention, proteins involved in the induction or regulation of the type I IFN response include, but are not limited to, cGAS, STING, TRIF,

TBK1, PSKepsilon, IRF3, TREX1, ¥PS34, ATG9a, DDX3, LC3, DDX41, IFI16,

MRE11, DNA-PK, RIG1, MDA5, LGP2, No. 8-1/MA¥8/cardif/¥18A, Trim25, Trim32, Trim56, riplet, TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, TLR3, TLR7, TLR9, DAI, IFI16, IFIX, MRE11, DDX41, LSml4A, LRRFIPl, DHX9, DHX36, DHX29, DHX15, Ku70, cyclic dinucleotide-forming enzymes (cyclic di-AMP, cyclic di-GMP cyclases and cyclic di-GAMP), such as WspR, DncV, DisA and DisA-like protein, CdaA, CdaS and cGAS or fragments thereof. Preferred proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of

- 22 044058 cGAS, STING, TRIF, TBK1,

PSKepsilon, IRF3, TREX1, VPS34, ATG9a, DDX3, LC3, DDX41, IFI16, MRE11, DNA-PK, RIG1, MDA5, LGP2, 1P8-1/MA¥8/cardif/¥18A, Trim25, Trim32, Trim56, riplet , TRAF2, TRAF3, TRAF5, TANK, IRF3, IRF7, IRF9, STAT1, STAT2, PKR, LSml4A, LRRFIP1, DHX29, DHX15 and cyclic dinucleotide-forming enzymes such as cyclic di-AMP, cyclic di-GMP and cyclic dinucleotide cyclases. -GAMP selected from the group consisting of WspR, DncV, DisA and DisA-like protein, CdaA, CdaS and cGAS or fragments thereof.

More preferred proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of cGAS (such as Uniprot. Q8N884 for a human protein), RIG1 (such as Uniprot. 095786 for a human protein), MDA5 (such as Uniprot. Q9BYX4 for human protein), IPS1/MAVS (such as Uniprot. Q7Z434 for human protein), IRF3 (such as Uniprot. Q14653 for human protein), IRF7 (such as Uniprot. Q92985 for human protein), IRF9 (such as Uniprot. Q00978 for human protein) and cyclic dinucleotide forming enzymes such as cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP cyclases selected from the group consisting of WspR (such as Uniprot. Q9HXT9 for P. aeruginosa protein), DncV (such as Uniprot. Q9KVG7 for the V. cholerae protein), DisA and DisA-like protein (such as Uniprot. Q812L9 for the B. cereus protein), CdaA (such as Uniprot. Q8Y5E4 for the L. monocytogenes protein), CdaS (such as Uniprot. 031854 or the constitutive active mutation L44F, such as in Seq ID No. 114 for the B. subtilis protein) and cGAS (such as Uniprot. Q8N884 for human protein) or fragments of these proteins.

IPS-1/MAVS/cardif/VISA designates a eukaryotic mitochondrial antiviral signaling protein containing an N-terminal CARD domain with Uniprot (www.uniprot.org) identifier Q7Z434 for the human sequence and Q8VCF0 for the mouse sequence. The terms IPS1/MAVS, MAVS/IPS-1 and MAVS are used interchangeably herein and refer to a eukaryotic mitochondrial antiviral signaling protein containing an N-terminal CARD domain with identifier (www.uniprot.org) Q7Z434 for the human sequence and Q8VCF0 for the mouse sequence.

In some embodiments, the heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of proteins containing a CARD domain or fragments thereof, and cyclic dinucleotide-forming enzymes, such as cyclic diAMP, cyclic di-GMP, and cyclic di-GAMP, or fragments thereof.

The fragment of heterologous proteins involved in the induction or regulation of the IFN response or the type I IFN response typically contains from 25 to 1000 amino acids, preferably from 50 to 600 amino acids, more preferably from 100 to 400 amino acids, even more preferably from 100 to 362 amino acids amino acids. In some embodiments, the fragment of heterologous proteins involved in the induction or regulation of the IFN response or the type I IFN response comprises an N-terminal fragment of the heterologous proteins involved in the induction or regulation of the IFN response or the type I IFN response, which typically contains from 25 to 1000 amino acids , preferably from 50 to 600 amino acids, more preferably from 100 to 400 amino acids, even more preferably from 100 to 362 amino acids, in particular from 100 to 246 amino acids or contains an N-terminal fragment of a heterologous protein involved in the induction or regulation of an IFN response or response A type I IFN that contains a deletion of the amino acid sequence contained between amino acid 1 and amino acid 160 of the N-terminal amino acids, preferably a deletion of the amino acid sequence containing N-terminal amino acids 1-59 or N-terminal amino acids 1-160, and wherein the N-terminal a heterologous protein fragment involved in the induction or regulation of the IFN response or type I IFN response typically contains from 25 to 1000 amino acids, preferably from 50 to 600 amino acids, more preferably from 100 to 400 amino acids, even more preferably from 100 to 362 amino acids amino acids.

Heterologous proteins containing a fragment of the CARD domain involved in the induction or regulation of the IFN response or the type I IFN response typically contain the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-500, preferably the amino acid sequence from N-terminal amino acid 1 to any of the N-terminal amino acids 100,400, more preferably, the amino acid sequence from the N-terminal amino acid 1 to any of the N-terminal amino acids 100-300, more preferably, the amino acid sequence from the N-terminal amino acid 1 to any of the N-terminal amino acids 100 -294, more preferably, the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-246.

In some embodiments, heterologous proteins containing a fragment of a CARD domain involved in the induction or regulation of an IFN response or a type I IFN response comprise an amino acid sequence selected from the group consisting of an amino acid sequence comprising at least an N-terminal amino acid 1 and no more than N-terminal amino acid 294,

- 23 044058 amino acid sequence containing at least N-terminal amino acid 1 and no more than N-terminal amino acid 246, amino acid sequence containing at least N-terminal amino acid 1 and no more than N-terminal amino acid 245, amino acid sequence, containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 229, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 228, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 218, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 217, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 100 and an amino acid sequence comprising at least N-terminal amino acid 1 and no more than N-terminal amino acid 101, more particularly, an amino acid sequence selected from the group consisting of an amino acid sequence containing at least N-terminal amino acid 1 and no more than the N-terminal amino acid 245, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 228, an amino acid sequence containing at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 217 and an amino acid sequence comprising at least N-terminal amino acid 1 and no more than N-terminal amino acid 100 of the human CARD domain.

In some preferred embodiments, heterologous proteins comprising a fragment of a CARD domain involved in the induction or regulation of an IFN response or a type I IFN response comprise an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 294 , amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 246, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 245, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 229, amino acid sequence from N -terminal amino acid 1 to N-terminal amino acid 228, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 218, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 217, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 100, the amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 101, more particularly, an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 245, amino acid sequence N-terminal amino acid 1 to N-terminal amino acid 228, amino acid sequence N-terminal amino acid 1 to N-terminal amino acid 217, and amino acid sequence N-terminal amino acid 1 to N-terminal amino acid 100 of the human CARD domain.

A fragment of cyclic dinucleotide-forming enzymes, such as cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP cyclases, typically contains the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-600, preferably the amino acid sequence from N-terminal amino acid 50 to any of N-terminal amino acids 100-550, more preferably the amino acid sequence from N-terminal amino acid 60 to any of N-terminal amino acids 100-530, in particular the amino acid sequence from N-terminal amino acid 60 to N-terminal amino acid 530, the amino acid sequence from N-terminal amino acid 146 to N-terminal amino acid 507, or the amino acid sequence from N-terminal amino acid 161 to N-terminal amino acid 530, more specifically, the amino acid sequence from N-terminal amino acid 161 to N -terminal amino acid 530 of human cGAS. In some embodiments, the cGAS fragment comprises, in particular, an amino acid sequence selected from the group consisting of an amino acid sequence comprising at least the N-terminal amino acid 60 and no more than the N-terminal amino acid N-terminal amino acid 422, an amino acid sequence comprising at least the N-terminal amino acid 146 and no more than the N-terminal amino acid N-terminal amino acid 507, and an amino acid sequence comprising at least the N-terminal amino acid 161 and no more than the N-terminal amino acid N-terminal amino acid 522. B In some embodiments, the cGAS fragment comprises, more specifically, an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 60 to N-terminal amino acid 422, the amino acid sequence from N-terminal amino acid 146 to N-terminal amino acid 507, and sequences from N-terminal amino acid 161 to N-terminal amino acid 522.

In a more preferred embodiment, the heterologous protein involved in the induction or regulation of the type I IFN response is selected from the group consisting of RIG1, MDA5 and MAVS/IPS-1 containing a CARD domain, or fragments thereof, and cGAS and a fragment thereof, in particular , selected from the group, with

- 24 044058 consisting of RIG1 containing a CARD domain and a fragment thereof, MAVS/IPS-1 containing a CARD domain and a fragment thereof, and cGAS and a fragment thereof. Fragments of these proteins are particularly preferred. In this more preferred embodiment, the CARD domain-containing RIG1, MDA5, and MAVS/IPS-1 comprise a naturally occurring CARD domain or domains and additionally the C-terminal amino acids after the naturally occurring CARD domain or domains comprising a naturally occurring helicase domain in in the case of RIG-1 or a fragment thereof, preferably a fragment containing 1-500, more preferably 1-250, even more preferably 1-150 amino acids, wherein the naturally occurring helicase domain or fragment thereof is not functional, i.e. does not bind to the CARD domain, or contain in the 5'-3' direction the C-terminal sequence in the case of MAVS/IPS-1 or a fragment thereof, preferably a fragment containing 1-500, more preferably 1-250, even more preferably, 1-150 amino acids. In these embodiments, the cGAS and fragment thereof typically comprise a naturally occurring synthase domain (N-Tase core and C-terminal domain; amino acids 160-522 of human cGAS as described in publication ⁶⁵ and such as Uniprot. Q8N884 for human protein), preferably cGAS and its fragment contains a naturally occurring synthase domain, but contains a deletion of part or all of the N-terminal domain, preferably a deletion of the entire N-terminal helical extension (N-terminal helical extension; amino acids 1-160 of human cGAS, as described in publication ⁶⁵ , and such as Uniprot. Q8N884 for human protein). The deletion of part or all of the N-terminal domain is preferably a deletion of amino acids 1-59.

In some embodiments, the 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 (such as RIG1 and MDA5) and a fragment thereof, other CARD domain-containing proteins , involved in antiviral signaling and induction of type I IFNs (such as MAVS/IPS-1) and their fragments, and enzymes that form cyclic dinucleotides, such as cyclases cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP, selected from the group consisting of WspR, DncV, DisA and DisA-like protein, CdaA, CdaS and cGAS and fragments thereof, which lead to stimulation of STING.

In some embodiments, the heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of RIG1, MDA5, LGP2, MAVS/IPS-1, WspR, DncV, DisA and DisA-like protein, CdaA, CdaS, and cGAS or fragments thereof are more preferably selected from the group consisting of RIG1, WspR, DncV, DisA-like protein and cGAS or fragments thereof.

In a more preferred embodiment, the protein involved in the induction or regulation of the type I IFN response is selected from the group consisting of RIG1, MDA5, MAVS/IPS-1, WspR, DncV, DisA and DisA-like protein, CdaA and cGAS, or a fragment thereof , even more preferably, selected from the group consisting of RIG1, MDA5, MAVS/IPS-1, WspR, DncV, DisA-like protein, CdaA and cGAS or a fragment thereof, in particular selected from the group consisting of RIG1, MAVS/ IPS-1 and cGAS or a fragment thereof. Fragments of these proteins are particularly preferred.

In this more preferred embodiment, the RIG1, MDA5, MAVS/IPS-1 fragment typically comprises the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-500, preferably the amino acid sequence from N-terminal amino acid 1 to any of N- terminal amino acids 100-400, more preferably, the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-300. In this more preferred embodiment, the RIG1 fragment comprises an amino acid sequence selected from the group consisting of an amino acid sequence comprising at least the N-terminal amino acid 1 and no more than the N-terminal amino acid 246, an amino acid sequence comprising at least the N-terminal amino acid 1 and no more than N-terminal amino acid 245, an amino acid sequence containing at least N-terminal amino acid 1 and no more than N-terminal amino acid 229, an amino acid sequence containing at least N-terminal amino acid 1 and no more N-terminal amino acid 228, an amino acid sequence comprising at least N-terminal amino acid 1 and no more than N-terminal amino acid 218, and an amino acid sequence containing at least N-terminal amino acid 1 and no more than N-terminal amino acid 217 ; and the MAVS/IPS-1 fragment contains an amino acid sequence selected from the group consisting of an amino acid sequence containing at least an N-terminal amino acid 1 and no more than an N-terminal amino acid 100 and an amino acid sequence containing at least an N-terminal amino acid 1 and no more than N-terminal amino acid 101.

In this more preferred embodiment, the RIG1 fragment comprises, more specifically, an amino acid sequence selected from the group consisting of an amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 246, an amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 245, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 229, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 228, amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 218 and amino acid

- 25 044058 sequence from N-terminal amino acid 1 to N-terminal amino acid 217; and the MAVS/IPS-1 fragment comprises, more specifically, an amino acid sequence selected from the group consisting of an amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 100 and an amino acid sequence from N-terminal amino acid 1 to N-terminal amino acid 101 In this more preferred embodiment, the cGAS fragment typically comprises the amino acid sequence from N-terminal amino acid 1 to any of N-terminal amino acids 100-600, preferably the amino acid sequence from N-terminal amino acid 50 to any of N-terminal amino acids 100-550 more preferably, the amino acid sequence from N-terminal amino acid 60 to any of N-terminal amino acids 100-530, in particular, the amino acid sequence from N-terminal amino acid 60 to N-terminal amino acid 530, the amino acid sequence from N-terminal amino acid 146 to N-terminal amino acid 507 or the amino acid sequence from N-terminal amino acid 161 to N-terminal amino acid 530, more specifically, the amino acid sequence from N-terminal amino acid 60 to N-terminal amino acid 530 or the amino acid sequence from N-terminal amino acid 161 to N- terminal amino acid 530 of human cGAS. In this more preferred embodiment, the cGAS fragment comprises, in particular, an amino acid sequence selected from the group consisting of an amino acid sequence comprising at least an N-terminal amino acid 60 and no more than an N-terminal amino acid, an N-terminal amino acid 422, an amino acid sequence containing at least an N-terminal amino acid 146 and no more than an N-terminal amino acid N-terminal amino acid 507, and an amino acid sequence containing at least an N-terminal amino acid 161 and no more than an N-terminal amino acid N-terminal amino acid 522 .

In this more preferred embodiment, the cGAS fragment more specifically comprises an amino acid sequence selected from the group consisting of the amino acid sequence from N-terminal amino acid 60 to N-terminal amino acid 422, the amino acid sequence from N-terminal amino acid 146 to N-terminal amino acid 507, amino acid sequence from N-terminal amino acid 161 to N-terminal amino acid 522.

In an even more preferred embodiment, the protein involved in the induction or regulation of the type I IFN response is selected from the group consisting of human RIG1 CARD1.245 domains (SEQ ID NO: 37), human RIG1 _{CARD1_228} domains (SEQ ID NO: 128) , human CARD1_ ₂₁₇ RIG1 domains (SEQ ID NO: 129), CARD1 domains. ₂₄₆ mouse RIG1 (SEQ ID NO: 38), CARD1-229 mouse RIG1 domains (SEQ ID NO: 110), CARD1_ domains ₂₁₈ mouse RIG1 (SEQ ID NO: 111), CARD1 domain. ₁₀₀ human MAVS (SEQ ID NO: 116), CARD1.101 domain mouse MAVS (SEQ ID NO: 130), N. vectensis cGAS (SEQ ID NO: 43), CGAS161. ₅₂₂ human (SEQ ID NO: 115), mouse cGAS ₁₄₆ - ₅₀₇ (SEQ ID NO: 131) and N. vectensis cGAS ₆₀ - ₄₂₂ (SEQ ID NO: 117).

In a particular preferred embodiment, the protein is involved in the induction or regulation of the type I IFN response, wherein the protein involved in the induction or regulation of the type I IFN response is selected from the group consisting of CARD1 domains. ₂₄₅ human RIG1 domains, (SEQ ID NO: 37), CARD1_ domains ₂₂₈ human RIG1 domains (SEQ ID NO: 128), human CARD1_ domains ₂₁₇ RIG1 (SEQ ID NO: 129), human CARD1_ domain ₁₀₀ MAVS (SEQ ID NO: 116) and cGAS _{161_522} humans (SEQ ID NO: 115) _.

In a more specific preferred embodiment, the protein involved in the induction or regulation of the type I IFN response is selected from the group consisting of CARD1 domains. ₂₄₅ human RIG1, CARD1 domains. 246 mouse RIG1, CARD1 domains. ₂₂₉ mouse RIG1, CARD1 domains. ₂₁₈ mouse RIG1, human MAVS1_ ₁₀₀ , N. vectensis cGAS, human cGAS ₁₆₁ - ₅₂₂ and N. vectensis cGAS ₆₀ - ₄₂₂ .

The RIG-I-like receptor (RLR) family contains proteins selected from the group consisting of RIG1, MDA5 and LGP2. Preferred heterologous proteins involved in the induction or regulation of the type I IFN response are the CARD domain-containing proteins RIG1 and MDA5, in particular the CARD domain-containing protein RIG1. Other CARD domain-containing proteins involved in the induction of type I IFN include proteins selected from the group consisting of MAVS/IPS-1.

In some preferred embodiments, the heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group of proteins comprising a RIG1 CARD domain, an MDA5 CARD domain, and/or a MAVS/IPS-1 CARD domain, and WspR, DncV, DisA, and DisA -like protein, CdaA, CdaS and cGAS and fragments thereof are preferably selected from the group of proteins containing the RIG1 CARD domain and/or the MAVS/IPS-1 CARD domain, and WspR, DncV, DisA and DisA-like protein, CdaA and cGAS or their fragments.

In some preferred embodiments, the heterologous proteins involved in the induction or regulation of the type I IFN response are selected from the group consisting of RIG1 CARD domain, MDA5 CARD domain, MAVS/IPS-1 CARD domain, WspR, DncV, DisA, and DisA-like protein , CdaA, CdaS and cGAS are more preferably selected from the group consisting of the RIG1 CARD domain, WspR, DncV, DisA-like protein and cGAS.

In some preferred embodiments, the heterologous proteins involved in the induction or regulation of the type I IFN response comprise one or more (e.g., two, three, or four)

- 26044058 CARD domains preferably comprise one or more (eg two, three or four) RIG1, MDA5 and/or MAVS/IPS-1 CARD domains, preferably RIG1 and/or MAVS/IPS-1. In a more preferred embodiment, the heterologous proteins involved in the induction or regulation of the type I IFN response contain RIG1 or MDA5 CARD domains, in particular RIG1.

In some embodiments, the heterologous proteins involved in inducing or regulating the type I IFN response are selected from the group consisting of a type I IFN response-inducing protein without enzymatic function or a type I IFN response-inducing protein with enzymatic function. The type I IFN response-inducing protein without enzymatic function included in the present invention typically contains at least one CARD domain, preferably two CARD domains. The CARD domain typically consists of an array of six to seven alpha helices, preferably an arrangement of six to seven antiparallel alpha helices with a hydrophobic core and an outer surface of charged residues. The type I IFN response-inducing protein with enzymatic function included in the present invention typically contains a cyclic dinucleotide-forming enzyme (cyclic di-AMP, cyclic di-GMP and cyclic diGAMP cyclases) or a domain thereof that leads to stimulation of STING, preferably , diadenylate cyclase (DAC), diguanylate cyclase (DGC) or GMP-AMP cyclase (GAC) or a domain thereof.

According to the present invention, proteins involved in apoptosis or regulation of apoptosis include, but are not limited to, 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, proteins 14-3-3, PKA, PKC, PI3K, Erk1/2, p90RSK, TRAF2, TRADD, FADD, Daxx, caspase8, caspase2, RIP, RAIDD, MKK7, JNK, FLIP, FKHR, GSK3, CDK and their inhibitors, such as the INK4 family (pl6(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, caspase9, caspase3, caspase6, caspase7, Smac/Diablo, Bok/Mtd, Bmf, Mcl-1 are used (S), LC8, PP2B, TRADD, Daxx, caspase8, caspase2, RIP, RAIDD, FKHR, CDK and their inhibitors such as INK4 family (p16(Ink4a), p15(Ink4b), p18(Ink4c), p19(Ink4d )), most preferably BIM, Bid, truncated Bid, FADD, caspase 3 (and its subunits), Bax, Bad, Akt, CDK and their inhibitors such as the INK4 family (p16(Ink4a), p15(Ink4b), p18 (Ink4c), p19(Ink4d)) ^11-13 . Additionally, proteins involved in apoptosis or regulation of apoptosis 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, caspase 8.

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

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, IAP family and Bfl-1.

Preferred are Bcl-2, Bcl-X1, Bcl-B, Bcl-W, Mcl-1, Ced-9, A1, NR13 and Bfl-1.

Inhibitors of anti-apoptosis pathways include proteins selected from the group consisting of Bad, Noxa and Cdc25A. Bad and Noxa are preferred.

Inhibitors of signaling or pro-survival pathways include proteins selected from the group consisting of PTEN, ROCK, PP2A, PHLPP, JNK, p38. Preferred are PTEN, ROCK, PP2A and PHLPP.

In some embodiments, the heterologous proteins involved in apoptosis or regulation of apoptosis are selected from the group consisting of B3-only proteins, caspases, and intracellular death receptor apoptosis control signaling proteins. BH3-only proteins are preferred. BNZ-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. The preferred ones are Bad, BIM, Bid and tBid, in particular tBid.

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, caspase 10. Preferred are caspase 3, caspase 8 and caspase 9 .

- 27 044058

Intracellular signaling proteins for the control of apoptosis by death receptors 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, families 14-3-3, FLIP, DFF40 and 45,

REA-15, SODD. FADD and TRADD are preferred.

In some embodiments, two heterologous proteins involved in apoptosis or the regulation of apoptosis are contained in a gram-negative bacterial strain, wherein one protein is a proapoptotic protein and the second protein is an inhibitor of pathways that prevent apoptosis, or wherein one protein is a proapoptotic protein and the second protein is an inhibitor of signaling or pro-survival pathways.

Proapoptotic proteins included in the present invention typically contain an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices, and typically contain at least one of BH1, BH2, BH3 or BH4 domains, preferably containing at least one BH3 domain. Typically, the proapoptotic proteins included in the present invention do not have enzymatic activity.

Antiapoptotic proteins included in the present invention typically contain an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices, and contain a combination of different BH1, BH2, BH3 and BH4 domains, preferably a combination of different BH1, BH2, BH3 and BH4 domains, in which there is a BH1 and BH2 domain, more preferably BH4-BH3-BH1-BH2, BH1-BH2, BH4-BH1-BH2 or BH3-BH1-BH2 (from N- to C-terminus). Additionally also included are proteins containing at least one BIR domain.

Inhibitors of anti-apoptosis pathways included in the present invention typically contain an alpha helical structure, preferably a hydrophobic helix surrounded by amphipathic helices, and typically contain a single BH3 domain.

Each of the BH1, BH2, BH3 or BH4 domains is typically from about 5 to about 50 amino acids in length. Thus, in some embodiments, the heterologous proteins involved in apoptosis or regulation of apoptosis are selected from the group consisting of heterologous proteins involved in apoptosis or regulation of apoptosis that are from about 5 to about 200, preferably from about 5 to about 150 , more preferably from about 5 to about 100, most preferably from about 5 to about 50, in particular from about 5 to about 25 amino acids in length.

In some embodiments, a gram-negative bacterial strain of the present invention comprises a nucleotide sequence encoding two domains of heterologous proteins involved in apoptosis or regulation of apoptosis, preferably two repeat, more preferably two identical repeat domains of a protein involved in apoptosis or regulation of apoptosis, or two domains of different proteins involved in apoptosis or regulation of apoptosis, most preferably two identical repeat domains of a protein involved in apoptosis or regulation of apoptosis. In some embodiments, a gram-negative bacterial strain of the present invention comprises a nucleotide sequence encoding two domains of heterologous proteins involved in apoptosis or regulation of apoptosis, one being a pro-apoptotic protein domain and the other being a protein domain that is an inhibitor of pathways that prevent apoptosis. , or wherein one is a pro-apoptotic protein domain and the other domain is a protein domain that is an inhibitor of signaling pathways or pro-survival pathways.

A particularly preferred heterologous protein is the BH3 domain of the apoptosis inducer tBID, more specifically a BH3 domain comprising a sequence selected from the group consisting of SEQ ID NO: 29-32, preferably SEQ ID NO: 31 or SEQ ID NO: 32.

Equally preferred is the BH3 domain of the apoptosis regulator BAX, more particularly a BAX domain comprising a sequence selected from the group consisting of SEQ ID NOs: 33-36, preferably SEQ ID NO: 35 or SEQ ID NO: 36. In SEQ ID NO includes human and mouse sequences, but the BH3 tBID and BAX domains of all other species are included equally.

In some embodiments, the heterologous protein repeat domains are a BH3 domain, preferably the tBID apoptosis inducer BH3 repeat domains, more preferably the tBID apoptosis inducer BH3 repeat domains contained in SEQ ID NO: 29-32 or SEQ ID NO: 25, or SEQ ID NO: 19, even more preferably, two BH3 repeat domains of the apoptosis inducer tBID, most preferably, two BH3 repeat domains of the tBID apoptosis inducer contained in SEQ ID NOs: 29-32, or SEQ ID NO: 25, or SEQ ID NO: 19, in particular, the two BH3 repeat domains of the apoptosis inducer tBID contained in the sequence SEQ ID NO: 27. Thus, in a preferred embodiment, the gram-negative bacterial strain and/or vector of the present invention contains a second DNA sequence encoding two BH3 repeat domains, more preferably, two BH3 repeat domains of the apoptosis inducer tBID. The two repeat domains may be connected by a linker of 1-30 amino acids in length, preferably 2-15 amino acids in length, more preferably 3-10 amino acids in length.

- 28 044058

In some embodiments, the two or more domains of the different heterologous proteins are heterologous protein domains that belong to the same functional class of proteins, preferably, the different heterologous proteins of the two or more domains are different heterologous proteins from a class of proteins involved in apoptosis or the regulation of apoptosis. In a preferred embodiment, the two or more domains of different heterologous proteins are the BH3 domain of the apoptosis inducer tBID and the BH3 domain of the apoptosis regulator BAX, particularly the BH3 fusion domains contained in SEQ ID NOs: 24 and 28. The two domains of different heterologous proteins may be connected by a linker of 1-30 amino acids in length, preferably 2-15 amino acids in length, more preferably 3-10 amino acids in length.

Another particularly preferred heterologous protein is a domain of a protein involved in the induction or regulation of a type I IFN response, more specifically a RIG1 CARD domain containing a sequence selected from the group consisting of SEQ ID NO: 37, 38, 110, 111, 128, 129, an MDA5 CARD domain containing a sequence selected from the group consisting of SEQ ID NO: 44-47, 112, 113, preferably SEQ ID NO: 112 or 113, or a MAVS/IPS-1 CARD domain containing a sequence selected from the group consisting of SEQ ID NO: 116, 48-49, preferably SEQ ID NO: 116, full-length cGAS such as N. vectensis cGAS (SEQ ID NO: 43), human CGAS ₁₆₁₅₂₂ (SEQ ID NO: 115) , N. vectensis cGAS _60-422 (SEQ ID NO: 117) or mouse cGAS _146-507 (SEQ ID NO: 131). Most specifically, a RIG1 CARD domain protein consisting of a sequence selected from the group consisting of SEQ ID NO: 37, 38, 110, 111, 128, 129, a CARD domain protein consisting of MAVS/IPS-1 containing a sequence selected from the group consisting of SEQ ID NO: 116, 48-49, preferably SEQ ID NO: 116, and full-length cGAS such as N. vectensis cGAS (SEQ ID NO: 43), human CGAS161-522 (SEQ ID NO: 115) , N. vectensis CGAS60-422 (SEQ ID NO: 117) or mouse CGAS146-507 (SEQ ID NO: 131).

In some embodiments, the heterologous proteins are a prodrug converting enzyme. In these embodiments, the recombinant virulence-attenuated gram-negative bacterial strain expresses, preferably expresses and secretes, a prodrug-converting enzyme. A prodrug converting enzyme as defined herein includes enzymes that convert non-toxic prodrugs into a toxic drug, preferably enzymes selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase, beta-galactosidase, carboxylesterases, nitroreductases, carboxypeptidases and beta-glucuronidases, more preferably enzymes selected from the group consisting of cytosine deaminase, purine nucleoside phosphorylase, thymidine kinase and beta-galactosidase.

The term protease cleavage site as used herein refers to a specific amino acid motif in an amino acid sequence, for example, in the amino acid sequence of a protein or fusion protein, which is cleaved by a specific protease recognizing the amino acid motif. For an overview, see publication ¹⁴ . Examples of protease cleavage sites are amino acid motifs that are cleaved by a protease selected from the group consisting of enterokinase (light chain), enteropeptidase, PreScission protease, human rhinovirus (HRV 3C) protease, TEV protease, TVMV protease, factor Xa protease, and thrombin. The following amino acid motifs are recognized by the corresponding proteases:

Asp-Asp-Asp-Asp-Lys: enterokinase (light chain)/enteropeptidase,

Leu-Glu-Val-Leu-Phe-Gln/Gly-Pro: PreScission protease/human rhinovirus protease (HRV 3C), Glu-Asn-Leu-Tyr-Phe-Gln-Ser and modified motifs based on, Glu-X-X- Tyr-X-Gln-Gly/Ser (where χ is any amino acid), recognized by the TEV (tobacco etch virus) protease,

Glu-Thr-Val-Arg-Phe-Gln-Ser: TVMV protease,

Ile-(Glu or Asp)-Gly-Arg. protease factor Xa,

Leu-Val-Pro-Arg/Gly-Ser: thrombin.

Protease cleavage sites herein include ubiquitin. Thus, in some preferred embodiments, ubiquitin is used as a protease cleavage site, i.e. nucleotide sequence encodes ubiquitin as a protease cleavage site that can be cleaved by specific ubiquitin-processing proteases at the N-terminal site, for example, which can be cleaved by specific ubiquitin-processing proteases called deubiquitinating enzymes at the N-terminal site endogenously in the cell into which it was delivered fusion protein. Ubiquitin is processed at the C-terminus by a group of endogenous ubiquitin-specific C-terminal proteases (deubiquitinating enzymes, DUBs). The degradation of ubiquitin by DUB is believed to occur at the very C-terminus of ubiquitin (after G76).

The individual, subject, or patient is a vertebrate. In certain embodiments, the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (eg, mice and rats). In preferred embodiments, the subject is a human.

The term mutation is used herein as a general term and includes single base pair and multiple base pair changes. Such mutations may include substitutions, frameshift mutations, deletions, insertions and truncations.

The term nuclear localization signal as used herein refers to an amino acid sequence that marks a protein for import into the nucleus of a eukaryotic cell and preferably includes a viral nuclear localization signal, such as the NLS derived from SV40 large T antigen (PPKKKRKV).

The term multiple cloning site as used herein refers to a short DNA sequence containing multiple restriction sites for digestion by restriction endonucleases such as

Acll, Hindlll, SspI, MluCI, Tsp509I, Pcil, Agel, BspMI, BfuAI, SexAI, MluI, BceAI, HpyCH4IV, HpyCH4III, Bael, BsaXI, AflIII, Spel, Bsrl, BmrI, Bglll, Afel, Alul, Stul, Seal, Clal, BspDI, PI-SceI, Nsil, Asel, Swal, CspCI, Mfel, BssSI, BmgBI, Pmll, Dralll, Alel, EcoP15I, PvuII, AlwNI, BtsIMutl, TspRI, Ndel, Nlalll, CviAII, Fatl, MslI, FspEI, Xcml, BstXI, PflMI, BccI, Ncol, BseYI, Faul, Smal, Xmal, TspMI, Nt.CviPII, LpnPI, Acil, SacII, BsrBI, MspI, Hpall, ScrFI, BssKI, StyD4I, BsaJI, BslI, Btgl, Neil, Avril, Mnll, BbvCI, Nb.BbvCI, Nt.BbvCI, Sbfl, BpulOI, Bsu36I, EcoNI, HpyAV, BstNI, PspGI, Styl, Bcgl, Pvul, BstUI, EagI, Rsrll, BsiEI, BsiWI, BsmBI, Hpy99I, MspAH, MspJI, SgrAI, Bfal, BspCNI, Xhol, Earl, Acul, PstI, Bpml, Ddel, Sfcl, AflII, BpuEI, Smll, Aval, BsoBI, MboII, BbsI, XmnI, BsmI, Nb.BsmI, EcoRI, Hgal, Aatll, Zral, TthllllPflFI, PshAI, AhdI, DrdI, Eco53kl, SacI, BseRI, Piel, Nt.BstNBI, Mlyl, Hinfl, EcoRV, Mbol, Sau3AI, DpnIIBfuCI, Dpnl, BsaBI, Tfil, BsrDI, Nb.BsrDI, Bbvl, BtsI, Nb.BtsI, BstAPI, SfaNI, SphI, NmeAIII, Nael, NgoMIV, Bgll, AsiSI, BtgZI, HinPlI, Hhal, BssHII, Notl, Fnu4HI, Cac8I, Mwol, Nhel, Bmtl, SapI, BspQI, Nt.BspQI, BlpI, Tsel, ApeKI, Bspl286I, Alwl, Nt.AlwI, BamHI, FokI, BtsCI, Haell, Phol, Fsel, Sfil, Nari, KasI, Sfol, PluTI, Asci, Ecil, BsmFI, Apal, PspOMI, Sau96I, NlalV, Kpnl, Acc65I, Bsal, HphI, BstEII, Avail, BanI, BaeGI, BsaHI, Banll, Rsal, CviQI, BstZ17I, BciVI, Sall, Nt.BsmAI, BsmAI, BcoDI, ApaLI, Bsgl, AccI, Hpyl66II, Tsp45I, Hpal, Pmel, Hindi, BsiHKAI, Apol, NspI, BsrFI, BstYI, Haell, CviKI-1, EcoO109I, PpuMI, I-Ceul, SnaBI, I-Scel, BspHI, BspEI, Mmel, TaqaI, Nrul, Hpyl88I, Nru188Sh, Xbal, Bell, HpyCH4V, FspI, PI-PspI, MscI, BsrGI, Msel, PacI, Psil, BstBI, Dral, PspXI, BsaWI, BsaAI, Eael, preferably, Xhol, Xbal, Hindllll, Ncol, Notl, EcoRI, EcoRV, BamHI, Nhel, SacI, Sall, BstBI.

The term multiple cloning site as used herein also refers to a short DNA sequence used for recombination events, such as in the Gateway cloning strategy, or for methods such as Gibbson assembly or TOPO cloning.

The term wild-type strain or wild-type gram-negative bacterial strain as used herein refers to a naturally occurring variant or naturally occurring variant containing genetic modifications allowing the use of vectors, such as deletion mutations in restriction endonucleases or antibiotic resistance genes. These strains contain chromosomal DNA, and also in some cases (for example, Y. enterocolitica, S. flexneri) an unmodified virulence plasmid.

The term wild-type Yersinia strain as used herein means a naturally occurring variant (such as Y. enterocolitica E40) or a naturally occurring variant containing genetic modifications allowing the use of vectors, such as deletion mutations in restriction endonucleases or antibiotic resistance genes (such as Y. enterocolitica MRS40, an ampicillin-sensitive derivative of Y. enterocolitica E40) These strains contain chromosomal DNA as well as an unmodified virulence plasmid (called pYV). Y. enterocolitica subspecies palearctica refers to low pathogenic strains of Y. enterocolitica that are distinct from the more virulent strains of enterocolitica subspecies ^15,16 . In Y. enterocolitica there is no subspecies palearctica, compared to Y.

- 30 044058 enterocolitica subspecies enterocolitica, high-pathogenicity island (HPI). The

HPI encodes an iron siderophore called yersiniabactin ¹⁷ . The absence of yersiniabactin in Y. enterocolitica subspecies palearctica makes the subspecies less pathogenic and dependent on inducible systemically available iron for persistent infection, for example in the liver or spleen ¹⁷ . Iron may become available to the bacterium in the individual, for example, by pre-administration of deferoxamine, an iron chelator used to treat iron overload in patients

The term contain is usually used to mean include, that is, allowing the presence of one or more properties or components.

The term approximately indicates a range of ±10% of the specified value. For example, the phrase approximately 200 includes ±10% of 200 or 180 to 220.

In one aspect, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain comprising a nucleotide molecule that contains 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, wherein the nucleotide sequence , encoding a delivery signal from a bacterial effector protein, is operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain further contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains the gene, encoding the specified endogenous protein required for growth, operably linked to the promoter.

In a further aspect, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain comprising a nucleotide molecule that contains 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, wherein the nucleotide sequence, encoding a delivery signal from a bacterial effector protein, operably linked to a promoter, and wherein the recombinant virulence-attenuated gram-negative bacterial strain additionally contains a modulation in RNA in the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type.

In a further aspect, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain comprising a nucleotide molecule that contains 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, wherein the nucleotide sequence, encoding a delivery signal from a bacterial effector protein, operably linked to a promoter, and wherein the heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response.

In a further aspect, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain as described herein for use in a method of treating cancer in a subject, the method comprising administering to the subject said recombinant virulence-attenuated Gram-negative bacterial strain, the recombinant virulence-attenuated bacterial strain a gram-negative strain of bacteria is administered in an amount that is sufficient to treat the subject. Preferably, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain as described herein for use in a method of treating cancer - a malignant solid tumor - in a subject, wherein the recombinant virulence-attenuated Gram-negative bacterial strain accumulates in a malignant solid tumor, and wherein said the method includes administering to a subject said recombinant virulence-attenuated gram-negative strain of bacteria, wherein the recombinant virulence-attenuated gram-negative strain of bacteria is administered in an amount that is sufficient to treat the subject.

In some embodiments, the recombinant virulence-attenuated Gram-negative bacterial strain is deficient in the production of at least one bacterial effector protein that is virulent against eukaryotic cells.

In one embodiment of the present invention, a recombinant virulence-attenuated gram-negative bacterial strain is transformed with a nucleotide molecule, for example, a vector that contains a 5' to 3' promoter;

a first nucleotide sequence encoding a delivery signal from a bacterial effector protein operably linked to said promoter;

a second nucleotide sequence encoding a heterologous protein fused in frame to the 3' end of said first nucleotide sequence.

- 31 044058

In one embodiment of the present invention, a recombinant virulence-attenuated Gram-negative bacterial strain is transformed with a nucleotide molecule, for example, a vector that contains, in the 5' to 3' direction, a first nucleotide sequence encoding a delivery signal or fragment thereof from a bacterial effector protein;

a second nucleotide sequence encoding a heterologous protein fused in frame to the 3' end of said first nucleotide sequence.

Preferably, the nucleotide sequence encoding the heterologous protein is flanked at the 3' end by a nucleotide sequence homologous to the nucleotide sequence of the chromosome or endogenous virulence plasmid at the 3' end of the delivery signal from the bacterial effector protein or a fragment thereof. More preferably, the nucleotide sequence flanking the homologous protein at the 3' end is homologous to a nucleotide sequence within 10 kb. on the chromosome or on an endogenous virulence plasmid at the 3' end of the delivery signal from a bacterial effector protein or fragment thereof. In particular, a given nucleotide sequence flanking a homologous protein at its 3' end is homologous to a nucleotide sequence within the same operon on the chromosome or on an endogenous virulence plasmid as the delivery signal from the bacterial effector protein, or a fragment thereof. In this embodiment, the transformation is typically carried out such that the fusion nucleotide sequence is inserted by homologous recombination on an endogenous virulence plasmid or chromosome, preferably an endogenous virulence plasmid, of a recombinant virulence-attenuated Gram-negative bacterial strain, and that the fusion nucleotide sequence is operably linked to the promoter of an endogenous virulence plasmid or chromosome, for example, a chromosomal pathogenicity island. Preferably, the fusion nucleotide sequence is operably linked to the promoter of the endogenous virulence plasmid. In this embodiment, the nucleotide sequence comprises a delivery signal or fragment thereof from a bacterial effector protein, preferably a fragment thereof, which enables homologous recombination at a homologous site on the chromosome or on an endogenous virulence plasmid, preferably on an endogenous virulence plasmid, which results in that the nucleotide sequence is placed in frame with the 3' end of the chromosome delivery signal or endogenous virulence plasmid, which is operably linked to the endogenous promoter.

In a further embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain or the recombinant virulence-attenuated Gram-negative bacterial strain is transformed with a nucleotide molecule, preferably a DNA nucleotide molecule, which contains a nucleotide sequence encoding a heterologous protein and a nucleotide sequence that is homologous or identical to the nucleotide a sequence encoding a delivery signal from a bacterial effector protein, or which is homologous or identical to a nucleotide sequence encoding a fragment of a delivery signal from a bacterial effector protein, wherein the delivery signal from a bacterial effector protein or a fragment thereof is encoded on the chromosome or on an endogenous virulence plasmid of the recombinant virulence attenuated gram-negative strain of bacteria. Preferably, a nucleotide sequence that is 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. More preferably, the nucleotide sequence encoding the heterologous protein is flanked at the 3' end by a nucleotide sequence homologous to the nucleotide sequence of the chromosome or endogenous virulence plasmid at the 3' end of the delivery signal from the bacterial effector protein, or a fragment thereof. Even more preferably, the nucleotide sequence flanking the homologous protein at the 3' end is homologous to a nucleotide sequence within 10 kb. on the chromosome or on an endogenous virulence plasmid at the 3' end of the delivery signal from a bacterial effector protein, or a fragment thereof. In particular, the nucleotide sequence flanking the homologous protein at the 3' end is homologous to the nucleotide sequence and is located within the same operon on the chromosome or on the endogenous virulence plasmid as the delivery signal from the bacterial effector protein, or a fragment thereof. In this embodiment, the transformation is typically carried out such that the nucleotide sequence encoding the heterologous protein is inserted on an endogenous virulence plasmid or chromosome of a recombinant virulence-attenuated Gram-negative bacterial strain, preferably on the endogenous virulence plasmid, at the 3' end of the delivery signal from the bacterial effector a protein encoded by a chromosome or an endogenous virulence plasmid, wherein the heterologous protein fused to the delivery signal is expressed and secreted.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains a deletion of the chromosomal gene encoding

- 32 044058 endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding the specified endogenous protein necessary for growth, operably linked to a promoter. Normally, the gene encoding an endogenous growth protein on an endogenous virulence plasmid encodes the same endogenous growth protein encoded by the deleted chromosomal gene. Preferably, the gene encoding an endogenous enzyme required for growth and located on an endogenous virulence plasmid contains its endogenous promoter and its endogenous transcription terminator. In case the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, the gene encoding the endogenous enzyme required for growth is located on the endogenous virulence plasmid pYV and preferably contains its endogenous promoter and its endogenous transcription terminator. The gene encoding the endogenous enzyme required for growth, the endogenous promoter and the endogenous transcription terminator are preferably located at a distance of 122 bp. in the 3'-5' direction from the start of orf155 (SycO) on an endogenous virulence plasmid, for example, on pYV. The gene encoding an endogenous enzyme required for growth, an endogenous promoter, and an endogenous transcription terminator typically replaces the insertion sequence found in pYVe40, the virulence plasmid of Y. enterocolitica strains MRS40 and E40, but not in pYVe227, the virulence plasmid of Y. enterocolitica W227O3 (Genbank: AF102990.1).

If the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, the endogenous virulence plasmid is pYV (plasmid of Yersinia Virulence, Yersinia virulence plasmid). In the event that the recombinant virulence-attenuated Gram-negative bacterial strain is a Salmonella strain, the endogenous location for insertion is one of the gene clusters called SpiI or SpiII (for the Salmonella pathogenicity island), a position at which the effector protein is encoded elsewhere or, in alternatively, one of the Salmonella virulence plasmids (SVPs).

Preferably, 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 inserted on an endogenous virulence plasmid at the native site of the bacterial effector protein, for example, at the native site of a virulence factor, preferably if the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, at the native site of YopE or another Yop (YopH, YopO, YopP, YopM, YopT), preferably at the native YopE site, or if the recombinant virulence-attenuated the gram-negative bacterial strain is a Salmonella strain at the native site of an effector protein encoded by SpiI, SpiII or encoded elsewhere, preferably at the native site of an effector protein encoded by SpiI or SpiII, more preferably at the native site of SopE or SteA. Preferably, the 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 operably linked to a native bacterial effector protein promoter present on an endogenous virulence plasmid, for example in the case where the recombinant is attenuated in terms of virulence, the gram-negative bacterial strain is a Yersinia strain, - with a native promoter from the Yersinia virulon gene, as indicated below, more preferably with a native YopE promoter or another Yop promoter (YopH, YopO, YopP, YopM, YopT), preferably with a native the YopE promoter, or if the recombinant virulence-attenuated Gram-negative bacterial strain is a Salmonella strain, with a native promoter from the SpiI or SpiII pathogenicity island or from an effector protein encoded elsewhere as specified below, more preferably with a native SopE promoter , InvB or SteA.

In one embodiment of the present invention, a recombinant virulence-attenuated Gram-negative bacterial strain comprises a deletion of a chromosomal gene encoding an endogenous growth protein and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding said endogenous growth protein operably linked with a promoter, wherein the gene encoding an endogenous protein necessary for growth is selected from a gene encoding an enzyme necessary for the production of an amino acid, a gene encoding an enzyme involved in the biosynthesis of peptidoglycan, a gene encoding an enzyme involved in the biosynthesis of LPS, a gene encoding an enzyme , involved in the synthesis of the nucleotide, and the gene encoding the translation initiation factor.

In one embodiment of the present invention, a recombinant virulence-attenuated Gram-negative bacterial strain comprises a deletion of a chromosomal gene encoding an endogenous growth protein and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding said endogenous growth protein operably linked with a promoter, and wherein the gene encoding an endogenous enzyme necessary for growth is a gene encoding an enzyme necessary for production

- 33 044058 amino acid production, wherein the enzyme necessary for the production of the amino acid is selected from the group consisting of aspartate beta-semialdehyde dehydrogenase (asd), glutamine synthetase (glnA), tryptophanyl tRNA synthetase (trpS) or serine hydroxymethyl transferase (glyA ) or transketolase 1 (tktA), transketolase 2 (tktB), ribulose phosphate 3-epimerase (rpe), ribose 5-phosphate isomerase A (rpiA), transaldolase A (talA), transaldolase B (talB), phosphoribosyl pyrophosphate synthase (prs) , ATP phosphoribosyltransferase (hisG), bifunctional histidine biosynthetic protein HisIE (hisI), 1-(5-phosphoribosyl)-5-[(5-phosphoribosylamino)methylidenamino]imidazole-4-carboxamide isomerase (hisA), subunit HisH imidazoleglycerol phosphate synthase (hisH) , HisF subunits of imidazoleglycerol phosphate synthase (hisF), bifunctional histidine biosynthesis 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), chorismate synthase (aroC), P protein (pheA), T protein (tyrA), aromatic amino acid aminotransferase (tyrB), phospho-2-dehydro-3-deoxyheptonaldolase (aroG), phospho-2-dehydro-3-deoxyheptonataldolase (aroH), phospho-2-dehydro-3-deoxyheptonataldolase (aroF), quinate/shikimate dehydrogenase (ydiB), ATP-dependent isoenzyme 6- phosphofructokinase 1 (pfkA), ATP-dependent isoenzyme 6-phosphofructokinase 2 (pfkB), fructose bisphosphate aldolase class 2 (fbaA), fructose bisphosphate aldolase class 1 (fbaB), triose phosphate isomerase (tpiA), pyruvate kinase I (pykF), pyruvate kinase II (pykA), glyceraldehyde-3phosphate dehydrogenase A (gapA), phosphoglycerate kinase (pgk), 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase (gpmA), 2,3-bisphosphoglycerate-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 I (sdaA), L-serine dehydratase 2 (sdaB), L-threonine dehydratase catabolic (tdcB), L -threonine dehydratase biosynthetic (ilvA), L-serine dehydratase (tdcG), 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- ribosylgomocysteinliasis (luxs), cystaione beta-Liazia, cystaion gamma-liazia, serine-hydroxymethyltransferase (Glya), glycinghydroxymethyltransferase, small subunit 3-izpropilmalatdehydase (LeuD), large subunit of 3-ampolm Latdehydratase (LEC), 3-Izopropilmalatdehydrogenase (LEUB ), L-threonine dehydratase biosynthetic (ilvA), large subunit of isoenzyme 3 acetolactate synthase (ilvI), small subunit of isoenzyme 3 acetolactate synthase (ilvH), small subunit of isoenzyme 1 acetolactate synthase (ilvN), small subunit of isoenzyme 2 acetolactate synthase (ilvM), ketol acid relative reductoisomerase (NADP(+)) (ilvC), dihydroxy acid dehydratase (ilvD), branched-chain amino acid aminotransferase (ilvE), bifunctional aspartokinase/homoserine dehydrogenase 1 (thrA), bifunctional aspartokinase/homoserine dehydrogenase 2 (metL), 2-isopropylmalate synthase (leuA ), glutamate pyruvate aminotransferase (alaA), aspartate aminotransferase (aspC), bifunctional aspartokinase/homoserine dehydrogenase 1 (thrA), bifunctional aspartokinase/homoserine dehydrogenase 2 (metL), lysine-sensitive aspartokinase 3 (lysC), aspartate semialdehyde dehydrogenase (asd), 2-keto-3-deoxy-galactonate aldolase (yagE), 4-hydroxy-tetrahydrodipicolinate synthase (dapA), 4-hydroxy-tetrahydrodipicolinate reductase (dapB), 2,3,4,5-tetrahydropyridine-2,6 -dicarboxylate N-succinyltransferase (dapD), succinyldiaminopimelate desuccinylase (dapE), diaminopimelate epimerase (dapF), putative lyase (yjhH), acetylornithine/succinyldiaminopimelate aminotransferase (argD), citrate synthase (gltA), aconitate hydratase B (acnB), aconite athydratase A (acnA), uncharacterized putative aconitate hydratase (ybhJ), isocitrate dehydrogenase (icd), aspartate aminotransferase (aspC), glutamate pyruvate aminotransferase (alaA), large chain glutamate synthase [NADPH] (gltB), small chain glutamate synthase [NADPH] (gltD) , glutaminesynthesis (GLNA), amino acid acetyltransferase (Arga), acetylglutamatkinase (Argb), n-acetyl-gamma-phosphate reductase (Argc), acetilornitin/succinopiminopimela-aminotransferase Lornin-deacilasis (Arge), chains f ornitinkarbamoiltransferase (argF), ornithine carbamoyltransferase chain I (argI), argininosuccinate synthase (argG), argininosuccinate lyase (argH), glutamate 5-kinase (proB), gamma-glutamyl phosphate reductase (proA), pyrroline-5-carboxylate reductase (proC), ornithine cyclodeaminase, leucine-tRNA ligase (leuS), glutamine-tRNA ligase (glnS), serine-tRNA ligase (serS), glycine-tPFIK ligase beta subunit (glyS), glycine-tRNA ligase alpha subunit (glyQ), tyrosine-tRNA ligase (tyrS) , threonine tRNA ligase (thrS), phenylalanine tRNA ligase alpha subunit (pheS), phenylalanine tRNA ligase beta subunit (pheT), arginine tRNA ligase (argS), histidine tRNA ligase (hisS), valine tRNA ligase (valS), alanine-tRNA ligase (alaS), isoleucine-tRNA ligase (ileS), proline-tRNA ligase (proS), cysteine-tRNA ligase (cysS), asparagine-tRNA ligase (asnS), aspartate-tRNA ligase ( aspS), glutamate-tRNA ligase (gltX), tryptophan-tRNA ligase (trpS), glycine-tRNA ligase beta subunit (glyS), methionine-tRNA ligase (metG), lysine-tRNA ligase (lysS). The preferred enzymes required for amino acid production are

- 34 044058 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, 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, most preferred is asd .

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain further comprises a modulation in a temperature-sensitive region of RNA in the 3'-5' direction of a gene encoding an endogenous DNA-binding protein such as AraC. Modulation in the temperature-sensitive region of the RNA in the direction 3'-5' from the gene encoding the endogenous DNA-binding protein of the AraC type can be a deletion, insertion or substitution in the temperature-sensitive region of the RNA. The deletion or insertion typically involves the deletion or insertion of one or more, preferably from about 30 to about 100 nucleotides, more preferably from about 40 to about 60 nucleotides. The substitution typically involves a substitution of one or more, preferably from about 3 to about 30 nucleotides, more preferably from about 3 to about 15 nucleotides. Preferably, the modulation in the temperature-sensitive region of the RNA in the 3'-5' direction from the gene encoding the endogenous AraC-type DNA binding protein is a deletion, preferably a deletion of about 30 to about 100 nucleotides, more preferably from about 40 to about 60 nucleotides nucleotides in the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type. The endogenous DNA-binding protein of the AraC type and the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding the DNA-binding protein of the AraC type are usually located on an endogenous virulence plasmid contained in a recombinant virulence-attenuated Gram-negative bacterial strain. The AraC type DNA binding protein is preferably selected from the group consisting of VirF, LcrF, MxiE, ExsA, PerA, HrpX, HrpB, GadX, HilC, HilD and InvF. More preferably, the AraC type DNA binding protein is selected from the group consisting of VirF and LcrF. In some embodiments, the recombinant virulence-attenuated gram-negative bacterial strain is Yersinia enterolitica, and the AraC-type DNA binding protein is VirF.

Preferably, modulation in the temperature-sensitive region of the RNA in the 3'-5' direction from the gene encoding the endogenous AraC-type DNA binding protein involves modulation that disrupts the RNA hairpin, preferably hairpin I, in the 3'-5' direction from the gene encoding endogenous DNA-binding protein of the AraC type. More preferably, the modulation in the temperature-sensitive region of the RNA in the 3'-5' direction from the gene encoding the endogenous AraC-type DNA binding protein involves a deletion that eliminates the RNA hairpin structure or portions thereof, preferably a portion of hairpin I, in the 3'-direction. 5' from the gene encoding the endogenous DNA-binding protein of the AraC type. A deletion that eliminates an RNA hairpin structure or portions thereof typically involves a deletion of about 30 to about 100 nucleotides, preferably about 40 to about 60 nucleotides. In some embodiments, the recombinant virulence-attenuated Gram-negative bacterial strain is Yersinia enterolitica, and the deletion includes a deletion of nucleotides at positions -111 to -57 in the 3' to 5' direction of the virF coding sequence (where -1 represents 1 base in the 3 '-5' from base A of the ATG start codon of the virF coding sequence).

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain is selected from the group consisting of the genera Yersinia, Escherichia, Salmonella, and Pseudomonas. In one embodiment, the recombinant virulence-attenuated gram-negative bacterial strain is selected from the group consisting of the genera Yersinia and Salmonella. Preferably, the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, more preferably a Yersinia enterocolitica strain. Most preferred is Yersinia enterocolitica E40 (0:9, biotype 2) ¹⁹ or its ampicillin-sensitive derivatives, such as Y. enterocolitica MRS40 (also called Y. enterocolitica subsp. palearctica MRS40), described in publication ²⁰ . Y. enterocolitica E40 and its derivative Y. enterocolitica MRS40, described in publication ²⁰ , are identical to Y. enterocolitica subspecies palearctica E40 and its derivative Y. enterocolitica subspecies palearctica MRS40, described in publications ^15,17,21 Also preferably recombinant virulence-attenuated Gram-negative the bacterial strain is a Salmonella strain, more preferably a Salmonella enterica strain. The most preferred is Salmonella enterica serovar Typhimurium SL1344, described in the Culture Collection of Public Health England (NCTC 13347).

- 35 044058

In some embodiments of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain is a strain that does not produce a siderophore, e.g., is deficient in the production of a siderophore, preferably does not produce a siderophore, e.g., is deficient in the production of any siderophore. Such a strain is, for example, Y. enterocolitica subspecies palearctica MRS40, described in publications ^15,17,20,21 , which does not produce yersiniabactin and which is preferred.

In one embodiment of the present invention, the bacterial effector protein delivery signal comprises a bacterial effector protein or an N-terminal fragment thereof, preferably a bacterial effector protein that is virulent against eukaryotic cells or an N-terminal fragment thereof.

In one embodiment of the present invention, the bacterial effector protein delivery signal is a bacterial T3SS effector protein, comprising a bacterial T3SS effector protein or an N-terminal fragment thereof, wherein the T3SS effector protein or an N-terminal fragment thereof may comprise a chaperone binding site. A T3SS effector protein or an N-terminal fragment thereof that contains a chaperone binding site is particularly suitable as a delivery signal in the present invention. Preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of

SopE, SopE2, SptP, YopE,

ExoS, SipA, SipB, SipD, SopA, SopB, SopD, IpgBl, IpgD, SipC, SifA, SseJ, Sse, SrfH, YopJ, AvrA, AvrBsT, YopT, YopH, YpkA, Tir, EspF, TccP2, IpgB2, OspF, Map, OspG, OspI, IpaH, SspHl, VopF, ExoS, ExoT, HopAB2, XopD, AvrRpt2, HopAOl, HopPtoD2, HopUl, GALA family proteins, AvrBs2, AvrDl, AvrBS3, YopO, YopP, YopE, YopM, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspCl, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpml, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD and AvrXv3.

More preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of

SopE, SptP, YopE, ExoS, SopB, IpgBl, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO,

YopP, YopE, YopM, YopT, of which the most preferred T3SS effector proteins or their N-terminal fragments are selected from the group consisting of W ¹ · ^So P ^E · ^So P ^B · ^γ "'' ^Os P ^F · ^Υο Ρ ^Η · ^Yo P°> ^Υο Ρ ^Ρ · ^Υο Ρ ^Ε · ^Υο Ρ ^Μ > ^Υο Ρ ^Τ · in particular, YopE or its N-terminal fragment.

Equally preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of

SopE, SopE2, SptP, SteA,

SipA, SipB, SipD, SopA, SopB, SopD, IpgBl, 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, HopAOl, HopUl, GALA family proteins, AvrBs2, AvrDl, YopO, YopP, YopE, YopT, EspG, EspH, EspZ, IpaA, IpaB, IpaC, VirA, IcsB, OspCl, OspE2, IpaH9.8, IpaH7.8, AvrB, AvrD, AvrPphB, AvrPphC, AvrPphEPto, AvrPpiBPto, AvrPto, AvrPtoB, VirPphA, AvrRpml, HopPtoD2, HopPtoE, HopPtoF, HopPtoN, PopB, PopP2, AvrBs3, XopD and AvrXv3.

Equally more preferred T3SS effector proteins or N-terminal fragments thereof are selected from the group consisting of

SopE, SptP,

SteA, SifB, SopB, IpgBl, IpgD, YopJ, YopH, EspF, OspF, ExoS, YopO, YopP, YopE,

YopT, among which the equally most preferred T3SS effector proteins or their N-terminal fragments are selected from the group consisting of

- 36 044058

IpgBl, SopE, SopB, SptP, SteA, SifB, OspF, IpgD, YopH, YopO, YopP, YopE and

YopT, in particular SopE, SteA or YopE or N-terminal fragments thereof, more particularly SteA or YopE or N-terminal fragments thereof, most particularly YopE or N-terminal fragment thereof.

In some embodiments, the bacterial effector protein delivery signal is encoded by a nucleotide sequence comprising a bacterial effector protein or an N-terminal fragment thereof, wherein the N-terminal fragment thereof comprises at least the first 10, preferably at least the first 20, more preferably at least the first 100 amino acids of the bacterial T3SS effector protein.

In some embodiments, the delivery signal from the bacterial effector protein is encoded by a nucleotide sequence comprising a bacterial T3SS effector protein or an N-terminal fragment thereof, wherein the bacterial T3SS effector protein or N-terminal fragment thereof comprises a chaperone binding site.

Preferred T3SS effector proteins or N-terminal fragments thereof that contain a chaperone binding site include the following combinations of a chaperone binding site and a T3SS effector protein or N-terminal fragment thereof

SycE-YopE, InvB-SopE, SicP-SptP, SycT-YopT, SycOYopO, SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF, SycD-YopB, SycDYopD. More preferred are SycE-YopE, InvB-SopE, SycT-YopT, SycO-YopO, SycN/YscB-YopN, SycH-YopH, SpcS-ExoS, CesF-EspF.

Most preferred is YopE or an N-terminal fragment thereof containing the SycE chaperone binding site, such as an N-terminal YopE effector protein fragment containing the N-terminal 138 amino acids of the YopE effector protein, referred to herein as YopE^ ₁₃₈ and represented by SEQ ID NO: 2, or SopE or an N-terminal fragment thereof containing an InvB chaperone binding site, such as an N-terminal fragment of a SopE effector protein containing the N-terminal 81 or 105 amino acids of the SopE effector protein, referred to herein as SopE _1-81 or SopE _{1- 105} , respectively, and presented in SEQ ID NO: 6 and 7.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, and the delivery signal from the bacterial effector protein comprises a YopE effector protein or an N-terminal portion thereof, preferably a Y. enterocolitica YopE effector protein or an N-terminal portion thereof . Preferably, the SycE binding site is contained in the N-terminal portion of the YopE effector protein. In this regard, the N-terminal fragment of the effector protein YopE may contain the N-terminal 12, 16, 18, 52, 53, 80 or 138 amino acids ^22,24 . Most preferred is an N-terminal YopE effector protein fragment containing the N-terminal 138 amino acids of the YopE effector protein, for example, as described by Forsberg and Wolf-Watz ²⁵ , referred to herein as YopE _1-138 and represented by SEQ ID NO: 2.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain is a Salmonella strain, and the bacterial effector protein delivery signal encoded by the nucleotide sequence comprises a SopE or SteA effector protein or an N-terminal portion thereof, preferably a SopE or SteA effector protein Salmonella enterica or its N-terminal part. Preferably, the chaperone binding site is contained in the N-terminal part of the SopE effector protein. In this regard, the N-terminal fragment of the SopE effector protein may contain the N-terminal 81 or 105 amino acids. Most preferred is full-length SteA (SEQ ID NO: 5) and an N-terminal SopE effector protein fragment containing the N-terminal 105 amino acids of the effector protein, for example, as described in SEQ ID NO: 7.

One skilled in the art will know methods for identifying effector protein polypeptide sequences that are capable of delivering the protein. For example, one such method is described in Sory et al. ¹⁹ . Briefly, polypeptide sequences, for example from various parts of the Yop proteins, 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 fusion protein into the cytosol of eukaryotic cells is marked by the appearance of cyclase activity in infected eukaryotic cells, which leads to the accumulation of cAMP. Using this approach, one skilled in the art can, if necessary, determine the minimum sequence requirement, i.e. for a contiguous amino acid sequence of the shortest length that is capable of delivering a protein, see, for example, publication ¹⁹ . Accordingly, preferred delivery signals of the present invention consist of at least the minimum amino acid sequence of a T3SS effector protein that is capable of delivering the protein.

In one embodiment, the present invention provides a recombinant virulence-attenuated Gram-negative bacterial strain that is deficient in

- 37 044058 tion of at least one bacterial effector protein, more preferably, which is deficient in the production of at least one bacterial effector protein, which is virulent against eukaryotic cells, even more preferably, which is deficient in the production of at least one effector a T3SS protein, most preferably, that is deficient in the production of at least one T3SS effector protein that is virulent against eukaryotic cells. In some embodiments, the recombinant virulence-attenuated Gram-negative bacterial strains are deficient in the production of at least one, preferably at least two, more preferably at least three, even more preferably at least four, in particular at least five, more specifically, at least six, most specifically, all bacterial effector proteins that are virulent against eukaryotic cells. In some embodiments, the recombinant virulence-attenuated Gram-negative bacterial strains are deficient in the production of at least one, preferably at least two, more preferably at least three, even more preferably at least four, in particular at least five, more specifically, at least six, most specifically, all functional bacterial effector proteins that are virulent against eukaryotic cells, such that the resulting recombinant virulence-attenuated Gram-negative bacterial strain produces fewer bacterial effector proteins or produces bacterial effector proteins to a lesser extent compared to a gram-negative strain of wild-type bacteria that is not attenuated in virulence, i.e. compared to a wild-type Gram-negative bacterial strain that normally produces bacterial effector proteins, or such that the resulting recombinant virulence-attenuated Gram-negative bacterial strain no longer produces any functional bacterial effector proteins that are virulent against eukaryotic cells.

According to the present invention, such a mutant gram-negative bacterial strain, i.e. such a recombinant virulence-attenuated Gram-negative bacterial strain that is deficient in the production of at least one bacterial effector protein, for example, that is deficient in the production of at least one bacterial effector protein that is virulent against eukaryotic cells, for example, such a mutant strain Yersinia can be obtained by introducing at least one mutation in at least one gene encoding an effector. Preferably, such effector encoding genes include Y°pE, YopH, YopO/YpkA, YopM, YopP/YopJ and YopT in the case of a Yersinia strain. Preferably, such effector encoding genes include

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, Ssel/SrfH, SopD, SopE, SopE2, SspHl, SspH2, PipB2, SifA, SopD2, SseJ, SseKl, SseK2, SseK3, SseL, SteC, SteA, SteB, SteD, SteE, SpvB, SpvC, SpvD, Srfl, SptP, if we are talking about the Salmonella strain. Most preferably, all genes encoding the effector are deleted. One skilled in the art can employ any number of standard techniques to obtain mutations in these T3SS effector genes. The manual by Sambrook et al. These methods are described in general terms. See Sambrook et al. for guidance. ²⁶ . According to the present invention, a mutation can be introduced in the promoter region of a gene encoding an effector, so that the expression of such an effector gene is eliminated.

A mutation can also be introduced into the coding region of a gene encoding an effector such that the catalytic activity of the encoded effector protein is eliminated. Effector protein catalytic activity generally refers to the function of the effector protein directed toward a target cell, i.e. toxicity. Such activity is regulated by catalytic motifs in the catalytic domain of the effector protein. Approaches for identifying the catalytic domain and/or catalytic motifs of an effector protein are well known to those skilled in the art. See, for example, publications ^27,28 .

Accordingly, one preferred mutation according to the present invention is a deletion of the entire catalytic domain. Another preferred mutation is a frameshift mutation in a gene encoding an effector such that the catalytic domain is not present in the protein product expressed from such a frameshift gene. The most preferred mutation is one that deletes the entire coding region of the effector protein. The present invention also provides other mutations, such as small deletions or base pair substitutions, that are introduced into the catalytic motifs of an effector protein and that result in disruption of the catalytic activity of the effector protein.

- 38 044058

Mutations that are obtained in the genes of functional bacterial effector proteins can be introduced into a particular strain using a variety of methods. One such method involves cloning the mutated gene into a suicide vector, which is capable of introducing the mutated sequence into the strain through allelic exchange. An example of such a suicide vector is described in publication ²⁹ .

Thus, mutations obtained in multiple genes can be sequentially introduced into a Gram-negative bacterial strain, resulting in a polymutant, for example, sixfold mutant, recombinant strain. The order in which these mutant sequences are introduced is not important. Under certain conditions, it may be desirable to obtain mutations in only some, but not all, effector genes. Accordingly, the present invention also provides polymutant Yersinia other than sixfold mutant Yersinia, such as doubly mutant, triply mutant, quadruple mutant and quintuple mutant strains. For the purpose of protein delivery, the secretion and translocation system of the present mutant strain must be intact.

A preferred recombinant virulence-attenuated Gram-negative bacterial strain of the present invention is a six-fold mutant strain of Yersinia in which all effector-encoding genes are mutated such that the resulting Yersinia no longer produces any functional effector proteins. This sixfold mutant strain of Yersinia is designated as AuopH,O,P,E,M,T for Y. enterocolitica. As an example, such a sixfold mutant can be obtained from Y. enterocolitica strain MRS40 to yield Y. enterocolitica MRS40 AuopH,O,P,E,M,T (also referred to herein as Y. enterocolitica subsp. palearctica MRS40 AuopH,O,P ,E,M,T or Y. enterocolitica AuopH,O,P,E,M,T), which is preferred. Y. enterocolitica MRS40 AuopH,O,P,E,M,T, which is deficient in the production of yersiniabactin, was described in publication WO02077249 and was deposited on ^September 24, 2001 under the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure in the Belgian Harmonized Collections of Microorganisms (HCCM); this strain was assigned the registration number LMG P-21013.

More preferred is Y. enterocolitica MRS40 AuopH,O,P,E,M,T, which contains a deletion on the endogenous virulence plasmid pYV eliminating the RNA hairpin structure or part thereof, such as deletion of Hairpin I in the 3' direction 5' from a gene encoding an endogenous DNA binding protein of the AraC type (AHairpinI-virF), such as Y. enterocolitica MRS40 AyopH,O,P,E,M,T AHairpinI-virF (also called Y. enterocolitica AyopH,O,P ,E,M,T AHairpinI-virF). Equally preferred is Y. enterocolitica MRS40 AuopH,O,P,E,M,T, which contains a deletion of the chromosomal gene encoding asd, and the endogenous virulence plasmid pYV containing a nucleotide sequence that contains the gene encoding asd, operably linked to promoter (pYV-asd) such as Y. enterocolitica MRS40 AyopH,O,P,E,M,T Aasd pYV-asd (also referred to herein as Y. enterocolitica AyopH,O,P,E,M,T Aasd pYV -asd). Particularly preferred is Y. enterocolitica MRS40 AyopH,O,P,E,M,T Aasd AHairpinI-virF pYV-asd, which contains both modifications described above (also referred to herein as Y. enterocolitica AyopH,O,P,E ,M,T Aasd AHairpinI-virF pYV-asd). Particularly preferred strains are Y. enterocolitica MRS40 AHairpinI-virF (also called Y. enterocolitica AHairpinI-virF), Y. enterocolitica MRS40 AHairpinI-virF ,O,P,E,M,T Aasd pYV-asd (also referred to herein as Y. Enterocolitica AyopH,O,P,E,M,T Aasd pYV-asd) or Y. enterocolitica MRS40 AyopH,O,P, E,M,T Aasd AHairpinI-virF pYV-asd (also referred to herein as Y. enterocolitica AyopH,O,P,E,M,T Aasd AHairpinI-virF pYV-asd) that are deficient in siderophore production, preferably do not produce siderophores, for example, are deficient in the production of any siderophore, as is the case with all strains of Y. enterocolitica subsp. palearctica. Thus, equally particularly preferred strains are Y. enterocolitica subsp. palearctica AyopH,O,P,E,M,T AHairpinI-virF (also called Y. enterocolitica subsp palearctica AyopH,O,P,E,M,T AHairpinI -virF), Y. enterocolitica subspecies palearctica AyopH,O,P,E,M,T Aasd pYV-asd, also referred to herein as Y. enterocolitica AyopH,O,P,E,M,T Aasd pYV-asd) or Y. enterocolitica subspecies palearctica AyopH,O,P,E,M,T Aasd AHairpinI-virF pYV-asd (also referred to herein as Y. enterocolitica AyopH,O,P,E,M,T Aasd AHairpinI-virF pYV-asd ).

Nucleotide molecules, such as vectors, that can be used according to the present invention to transform a gram-negative bacterial strain may depend on the gram-negative bacterial strains used, as is known to one skilled in the art. Nucleotide molecules that can be used according to the present invention include expression vectors (including synthetic or otherwise modified versions of endogenous virulence plasmids), chromosomal insertion or virulence plasmid insertion vectors, and nucleotide sequences, such as, for example, DNA fragments for chromosomal insertion or virulence plasmid inserts. Expression vectors that are suitable, for example, in strains of Yersinia, Escherichia, Salmonella or Pseudomonas are, for example, the plasmids pUC, pBad, pACYC, pUCP20 and pET. Vectors for chromosomal insertion or virulence plasmid insertion that are suitable, for example, in a Yersinia, Escherichia, Salmonella or Pseudomonas strain are, for example, pKNG101. DNA fragments for chromosomal insertion or insertion of a virulence plasmid indicate methods used, for example, in a strain of Yersinia, Escherichia, Salmonella or Pseudomonas, such as lambda-red genetic engineering. Vectors for chromosomal insertion or virulence plasmid insertion, or DNA fragments for chromosomal insertion or virulence plasmid insertion, can insert nucleotide sequences according to the present invention such that, for example, a nucleotide sequence encoding a heterologous protein is fused in frame to the 3' end of a nucleotide sequence encoding delivery signal from a bacterial effector protein, functionally linked to the endogenous promoter of a recombinant virulence-attenuated gram-negative bacterial strain. Thus, if a vector for chromosomal or virulence plasmid insertion or a DNA fragment for chromosomal insertion or virulence plasmid insertion is used, the endogenous promoter may be encoded on the endogenous bacterial DNA (chromosomal or plasmid DNA), and only the corresponding nucleotide sequence will be provided by the constructed vector for chromosomal or virulence plasmid insertion. insertion or insertion of a virulence plasmid or a DNA fragment for a chromosomal insertion or insertion of a virulence plasmid. Alternatively, if a chromosomal or virulence plasmid insertion vector or a nucleotide molecule is used, such as, for example, a nucleotide sequence for a chromosomal or virulence plasmid insertion, the endogenous promoter and delivery signal from the bacterial effector protein may be encoded on the endogenous bacterial DNA (chromosomal or plasmid DNA), and only a nucleotide molecule, such as, for example, a nucleotide sequence encoding a heterologous protein, will be provided by a vector for a chromosomal or virulence plasmid insertion, or a nucleotide molecule, such as, for example, a nucleotide sequence for a chromosomal or virulence plasmid insertion . Thus, it is not required that the promoter be necessarily contained in the vector used for the transformation of recombinant virulence-attenuated Gram-negative bacterial strains, i.e. The recombinant virulence-attenuated Gram-negative bacterial strains of the present invention can be transformed with a vector that does not contain a promoter.

In a preferred embodiment, the nucleotide molecule, for example, a vector according to the present invention, contains in the 5' to 3' direction a first nucleotide sequence encoding a delivery signal or fragment thereof from a bacterial effector protein;

a second nucleotide sequence encoding a heterologous protein fused in frame to the 3' end of said first nucleotide sequence.

A preferred vector, eg a preferred expression vector for Yersinia, is selected from the group consisting of pBad_Si_1 and pBad_Si_2. pBad_Si2 was constructed by cloning the SycE-YopE ₁ fragment. ₁₃₈ containing endogenous promoters for YopE and SycE, from purified pYV40 into the KpnI/HindIII site of pBad-MycHisA (Invitrogen). Additional modifications include removal of the NcoI/BgIII fragment of pBad-MycHisA by cleavage, treatment with Klenow fragment, and religation. Additionally, the following cleavage sites were added at the 3' end of YopE1_ ₁₃₈ : XbaIXhoI-BstBI-(HindIII). pBad_Si1 is similar to pBad_Si2 but encodes EGFP amplified from pEGFPC1 (Clontech) at the NcoI/BgIII site under the control of an arabinose-inducible promoter. Equally preferred is the use of modified versions of the endogenous Yersinia virulence plasmid pYV, encoding heterologous proteins as fusions to the T3SS signal sequence.

A preferred vector, for example a preferred expression vector for Salmonella, is selected from the group consisting of pSi_266, pSi_267, pSi_268 and pSi_269. Plasmids pSi_266, pSi_267, pSi_268 and pSi_269, containing the corresponding endogenous promoter and fragment SteA1_ ₂₀ (pSi_266), the full-length SteA sequence (pSi_267), fragment SopE1_ ₈₁ (pSi_268) or fragment SopE1_ ₁₀₅ (pSi_269), were amplified from genomic DNA of S. enterica SL1344 and cloned into the NcoI/KpnI site of pBadMycHisA (Invitrogen).

Nucleotide molecules, for example, the vectors of the present invention, may contain other sequence elements, such as a 3' termination sequence (including a stop codon and a poly-A sequence), or a drug resistance gene that allows selection of transformants receiving the present invention. vector.

Recombinant virulence-attenuated Gram-negative bacterial strains can be transformed with nucleotide molecules, such as the vectors of the present invention, in a variety of known ways. For purposes of the present invention, methods of transformation to introduce a vector include, but are not limited to, electroporation, calcium phosphate-mediated transformation, conjugation, or combinations thereof. For example, nucleotide molecules, e.g.

- 40 044058 vector, the first strain of bacteria can be transformed using a standard electroporation procedure. Such nucleotide molecules, such as a vector, can then be transferred from the first strain of bacteria to the desired strain through conjugation, a process also called mobilization. The transformant (i.e., gram-negative strains of bacteria that have captured the vector) can be selected, for example, using antibiotics. These techniques are well known in the art. See, for example, publication ¹⁹ .

According to the present invention, the promoter operably linked to the bacterial effector protein of the recombinant virulence-attenuated Gram-negative bacterial strain of the present invention may be the native T3SS effector protein promoter of the corresponding strain or a compatible bacterial strain, or a promoter used in expression vectors that are suitable, for example, in a strain of Yersinia, Escherichia, Salmonella or Pseudomonas, for example pUC and pBad. Such promoters are the T7 promoter, the Plac promoter or the arabinose inducible Ara-bad promoter. If the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, the promoter may be derived from the Yersinia virulon gene. The Yersinia virulon gene refers to genes on the pYV Yersinia plasmid whose expression is controlled by both temperature and contact with the target cell. Such genes include genes encoding elements of the secretion apparatus (Ysc genes), genes encoding translocators (YopB, YopD and LcrV), genes encoding control 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 proteins encoded by pYV, such as VirF and YadA.

In a preferred embodiment of the present invention, the promoter is the native promoter of a gene encoding a functional T3SS effector. If the recombinant virulence-attenuated Gram-negative bacterial strain is a Yersinia strain, the promoter is selected from any one of YopE, YopH, YopO/YpkA, YopM and YopP/YopJ. More preferably, the promoter is derived from YopE or SycE. Most preferred is the YopE promoter.

If the recombinant virulence-attenuated Gram-negative bacterial strain is a Salmonella strain, the promoter may be derived from the pathogenicity island SpiI or SpiII or from an effector protein encoded elsewhere. Such genes include genes encoding secretion apparatus elements, genes encoding translocators, genes encoding control elements, genes encoding T3SS effector chaperones, and genes encoding effectors, as well as other proteins encoded by SPI-1 or SPI-2. In a preferred embodiment of the present invention, the promoter is the native promoter of a gene encoding a functional T3SS effector. If the recombinant virulence-attenuated Gram-negative bacterial 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. In some embodiments, the promoter is an artificially inducible promoter, such as, for example, an arabinose inducible promoter, which is preferred. In this case, the bacteria are typically provided with arabinose, which then induces bacterial expression of the protein to be delivered.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains a nucleotide sequence encoding a protease cleavage site. The protease cleavage site is typically located on a nucleotide molecule that contains 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, between the nucleotide sequence encoding the heterologous protein and the nucleotide sequence encoding delivery signal. Obtaining a functional and generally useful cleavage site allows the delivery signal to be cleaved after translocation. Since the delivery signal may interfere with the proper localization and/or function of the translocated protein in target cells, the introduction of a protease cleavage site between the delivery signal and the protein of interest allows delivery of essentially native proteins into eukaryotic cells. Preferably, the protease cleavage site is an amino acid motif that is cleaved by a protease or its catalytic domains selected from the group consisting of enterokinase (light chain), enteropeptidase, PreScission protease, human rhinovirus 3C protease, TEV protease, TVMV protease, factor Xa protease, and thrombin, more preferably an amino acid motif that is cleaved by TEV protease. An equally preferred protease cleavage site is an amino acid motif that is cleaved by a protease or its catalytic domains selected from the group consisting of enterokinase (light chain), enteropeptidase, PreScission protease, human rhinovirus 3C protease, TEV protease, TVMV protease, protease factor Xa, ubiquitin-processing proteases called deubiquitinating enzymes, and thrombin. Most preferred is an amino acid motif that is cleaved by TEV protease or ubiquitin-processing protease. Thus, in a further embodiment of the present invention, the heterologous protein is cleaved from the delivery signal from the bacterial effector protein by a protease. Preferred cleavage methods are those in which:

a) the protease is translocated into a eukaryotic cell by a recombinant virulence-attenuated Gram-negative bacterial strain as described herein that expresses a fusion protein containing a delivery signal from a bacterial effector protein and the protease as a heterologous protein; or

b) the protease is expressed constitutively or transiently in the eukaryotic cell.

Typically, the recombinant virulence-attenuated Gram-negative bacterial strain used to deliver the desired protein into the eukaryotic cell and the recombinant virulence-attenuated Gram-negative bacterial strain that translocates the protease into the eukaryotic cell are different.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains an additional nucleotide sequence encoding a labeling molecule or an acceptor site for a labeling molecule. An additional nucleotide sequence encoding a labeling molecule or an acceptor site for a labeling molecule is typically fused to the 5' end or 3' end of a nucleotide sequence encoding a heterologous protein. The preferred labeling molecule or acceptor site for the labeling molecule is selected from the group consisting of enhanced green fluorescent protein (EGFP), coumarin, coumarin ligase acceptor site, resorufin, resorufin ligase acceptor site, tetracysteine motif used with the FlAsH dye. ReAsH (Life Technologies). Most preferred is resorufin and resorufin ligase acceptor site or EGFP. The use of a tagging molecule or an acceptor site for a tagging molecule will attach the tagging molecule to a heterologous protein of interest, which will then be delivered as such into the eukaryotic cell and enable tracking of the protein, for example, by live cell microscopy.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains an additional nucleotide sequence encoding a peptide tag. An additional nucleotide sequence encoding a peptide tag is typically fused to the 5' end or 3' end of a nucleotide sequence encoding a heterologous protein.

The preferred peptide tag is selected from the group consisting of a Myc tag, a His tag, a Flag tag, a HA tag, a Strep tag, or a V5 tag, or a combination of two or more tags from this group. Most preferred are a Myc tag, a Flag tag, a His tag, and a combination of Myc and His tags. The use of a peptide tag will provide the ability to monitor the tagged protein, for example, using immunofluorescence or Western blotting using antibodies against the tag. Additionally, the use of a peptide tag allows affinity purification of the desired protein after secretion into the culture supernatant or after translocation into eukaryotic cells, in both cases using a purification method suitable for the appropriate tag (e.g., metal chelate affinity purification when used with a His tag or purification on based anti-Flag antibody when used with a Flag tag).

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains an additional nucleotide sequence encoding a nuclear localization signal (NLS). An additional nucleotide sequence encoding a nuclear localization signal (NLS) is typically fused to the 5' end or 3' end of a nucleotide sequence encoding a heterologous protein, said additional nucleotide sequence encoding a nuclear localization signal (NLS). The preferred NLS is selected from the group consisting of the SV40 large T antigen NLS and its derivatives ³⁰ as well as other viral NLSs. Most preferred is the SV40 large T antigen NLS and its derivatives.

In one embodiment of the present invention, the recombinant virulence-attenuated Gram-negative bacterial strain contains a multiple cloning site. The multiple cloning site is typically located at the 3' end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein and/or at the 5' end or 3' end of a nucleotide sequence encoding a heterologous protein. The vector may contain one or more than one multiple cloning site. The preferred multiple cloning site is selected from the group of restriction enzymes consisting of XhoI, XbaI, HindIII, NcoI, NotI, EcoRI, EcoRV, BamHI, NheI, Sad, SalI, BstBI. Most preferred are XbaI, XhoI, BstBI and HindIII.

The fusion protein expressed by the recombinant virulence-attenuated Gram-negative bacterial strain of the present invention is also referred to as fusion protein

- 42 044058 or hybrid protein, i.e. a fusion protein or hybrid of a delivery signal and a heterologous protein. The fusion protein may also comprise, for example, a delivery signal and two or more different heterologous proteins.

The present invention provides methods for treating cancer in a subject, eg, treating malignant solid tumors, comprising delivering heterologous proteins, as described hereinabove, to cancer cells, eg, malignant solid tumor cells. Proteins can be delivered, i.e. translocate into a cancer cell, for example, into malignant solid tumor cells, during the administration of a recombinant virulence-attenuated Gram-negative strain of bacteria to a subject, or they can be delivered, i.e. translocate into a cancer cell, e.g., a malignant solid tumor cell, at a later time, e.g., after a recombinant virulence-attenuated Gram-negative bacterial strain reaches a cancer cell, e.g., a site of a malignant solid tumor, and/or reaches a cancer cell, e.g. , site of a malignant solid tumor, and replicates as described above. The delivery time can be controlled, for example, using a promoter, which is used for the expression of heterologous proteins in a recombinant virulence-attenuated Gram-negative bacterial strain. In the first case, the heterologous protein may be driven by a constitutive promoter or, more preferably, an endogenous promoter of a bacterial effector protein. In the case of delayed delivery of proteins, the heterologous protein can be driven by an artificially inducible promoter, such as an arabinose inducible promoter. In this case, after the bacteria have reached and accumulated in the desired area, arabinose will be administered to the subject. Arabinose then induces bacterial expression of the protein to be delivered.

Thus, in one embodiment, a method of treating cancer includes:

i) cultivating a recombinant virulence-attenuated gram-negative bacterial strain as described herein;

ii) administering to the subject said recombinant virulence-attenuated gram-negative bacterial strain by contacting the cancer cell with the recombinant virulence-attenuated gram-negative bacterial strain according to claim i), wherein a fusion protein that contains a delivery signal from a bacterial effector protein and a heterologous protein is expressed by the recombinant attenuated by virulence, a gram-negative strain of bacteria and translocates into a cancer cell; and optionally iii) cleaving the fusion protein such that the heterologous protein is cleaved from the delivery signal from the bacterial effector protein within the cancer cell, wherein the recombinant virulence-attenuated Gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject.

Cancer cells for delivery of heterologous proteins are typically cancer cells from cancer types selected from non-solid tumors selected from the group consisting of sarcoma, leukemia, lymphoma, multiple myeloma, central nervous system cancer types, and malignant solid tumors, which include, but are not limited to , an abnormal mass of cells that can originate from various types of tissues, such as the liver, colon, colon, rectum, skin, breast, pancreas, cervix, corpus uterus, bladder, gallbladder, kidney, larynx, lip , oral cavity, esophagus, ovary, prostate, stomach, testes, thyroid or lung, and thus includes malignant solid tumors of the liver, colon, colorectum, skin, breast, pancreas, cervix, body of the uterus, bladder, gall bladder, kidneys, larynx, lip, oral cavity, esophagus, ovary, prostate, stomach, testes, thyroid or lungs. Preferably, the cancer cells for delivery of heterologous proteins are malignant solid tumors.

Thus, in one preferred embodiment, the cancer is a malignant solid tumor, and the method includes:

i) cultivating a recombinant virulence-attenuated gram-negative bacterial strain as described herein;

ii) administering to the subject said recombinant virulence-attenuated gram-negative bacterial strain by contacting a malignant solid tumor cell with the recombinant virulence-attenuated gram-negative bacterial strain of claim i), wherein a fusion protein that contains a delivery signal from a bacterial effector protein and a heterologous protein is expressed a recombinant gram-negative strain of bacteria attenuated in virulence and translocated into the cell of a malignant solid tumor; and optionally iii) cleaving the fusion protein such that the heterologous protein is cleaved from the delivery signal from the bacterial effector protein within the malignant solid tumor cell, wherein the recombinant virulence-attenuated Gram-negative bacterial strain is administered in an amount that is sufficient to treat the subject.

In some embodiments, at least two fusion proteins, each comprising a delivery signal from a bacterial effector protein and a heterologous protein, are expressed by a recombinant virulence-attenuated Gram-negative bacterial strain and translocated into a eukaryotic cell, such as a cancer cell, by methods according to the present invention.

A recombinant virulence-attenuated Gram-negative bacterial strain can be cultured to express a fusion protein that contains a delivery signal from a bacterial effector protein and a heterologous protein, according to methods known in the art (for example, see the FDA publication, Bacteriological Analytical Manual (BAM) , chapter 8: Yersinia enterocolitica). Preferably, the recombinant virulence-attenuated Gram-negative bacterial strain can be cultured in brain heart extract broth, for example at 28°C. To induce expression of T3SS and, for example, YopE/SycE promoter-dependent genes, bacteria can be grown at 37°C. In one embodiment, a cancer cell, for example, a malignant solid tumor cell, is contacted with two recombinant virulence-attenuated Gram-negative bacterial strains according to claim i), wherein the first recombinant virulence-attenuated Gram-negative bacterial strain expresses a first fusion protein that contains a delivery signal from a bacterial effector protein and a first heterologous protein, and a second recombinant virulence-attenuated Gram-negative bacterial strain expresses a second fusion protein that contains a delivery signal from the bacterial effector protein and a second heterologous protein such that the first and second fusion proteins are translocated into a malignant solid tumor cell. This embodiment allows for co-infection of a cancer cell, for example a malignant solid tumor cell, with two strains of bacteria as a reliable method for delivering, for example, two different fusion proteins into individual cells to direct their functional interaction.

Those skilled in the art may also employ a variety of assays to determine whether delivery of the fusion protein has been successful. For example, the fusion protein can be detected by immunofluorescence using antibodies that recognize the fusion tag (such as the Myc tag). The determination may also be based on the enzymatic activity of the protein being delivered, for example, as in the assay described in publication ¹⁹ .

The present invention also provides a pharmaceutical composition comprising a recombinant virulence-attenuated Gram-negative bacterial strain as described herein, optionally containing a suitable pharmaceutically acceptable carrier. Thus, the present invention also provides a pharmaceutical composition comprising a recombinant virulence-attenuated Gram-negative bacterial strain as described herein for use in a method of treating cancer, for example, a malignant solid tumor in a subject.

Recombinant virulence-attenuated Gram-negative bacteria can be formulated for convenient and effective administration in an amount sufficient to treat a subject in the form of a pharmaceutical composition with a suitable pharmaceutically acceptable carrier. A unit dosage form of recombinant virulence-attenuated gram-negative bacteria or pharmaceutical composition to be administered may, for example, contain recombinant virulence-attenuated gram-negative bacteria in an amount of from about 10 ⁵ to about 10 ⁹ bacteria per ml, preferably from about 10 ⁶ to about 10 ⁸ bacteria per 1 ml, more preferably from about 10 ⁷ to about 10 ⁸ bacteria per 1 ml, most preferably about 10 ⁸ bacteria per 1 ml.

By an amount sufficient to treat the subject, or an effective amount, as used interchangeably herein, is meant an amount of bacterium or bacteria that is high enough to significantly beneficially modify the condition being treated, but low enough to avoid serious side effects ( with a reasonable benefit/risk ratio), within the bounds of sound medical judgment. The effective amount of bacterium will vary depending on the specific purpose desired, the age and physical condition of the subject being treated, the duration of treatment, the nature of the concomitant therapy and the particular bacterium used. An effective amount of bacteria will therefore be the minimum amount that will provide the desired effect. Typically, the subject is administered an amount of from about 10 ⁵ to about 10 ⁹ bacteria, such as from about 10 ⁵ to about 10 ⁹ bacteria/m ² of body surface, preferably from about 10 ⁶ to about 10 ⁸ bacteria, such as from about 10 ⁶ to about 10 ⁸ bacteria/m ² body surface, more preferably about 10 ⁷ to about 10 ⁸ bacteria, e.g. about 10 ⁷ to about 10 ⁸ bacteria/m ² body surface, most preferably 10 ⁸ bacteria, e.g. ⁸ bacteria/ ^m2 body surface.

A single dose of a recombinant virulence-attenuated Gram-negative bacterial strain for administration to a subject, e.g., a human, for the treatment of cancer, e.g., a malignant solid tumor, is typically from about 10 ⁴ to about 10 ¹⁰ bacteria,

- 44 044058 for example from about 10 ⁴ bacteria/m ² body surface to about 10 ¹⁰ bacteria/m ² body surface, preferably from about 10 ⁵ to about 10 ⁹ bacteria, for example from about 10 ⁵ to about 10 ⁹ bacteria/ m ² body surface, more preferably from about 10 ⁶ to about 10 ⁸ bacteria, for example from about 10 ⁶ to about 10 ⁸ bacteria/m ² body surface, even more preferably from about 10 ⁷ to about 10 ⁸ bacteria, for example , from about 10 ⁷ to about 10 ⁸ bacteria/m ² body surface, most preferably, 10 ⁸ bacteria, for example, 10 ⁸ bacteria/m ² body surface total recombinant virulence-attenuated Gram-negative bacteria.

Examples of substances that can act as pharmaceutical carriers are sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethylcellulose and cellulose acetates; powdered tragacanth; malt; gelatin; talc; stearic acids; magnesium stearate; calcium sulfate; calcium carbonate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil and cocoa bean oil; polyhydric alcohols such as propylene glycol, glycerin, sorbitol, mannitol and polyethylene glycol; agar; alginic acids; pyrogen-free water; isotonic saline solution; cranberry extracts and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations, such as, for example, vitamin C, estrogen and echinacea. Wetting agents and lubricants such as sodium lauryl sulfate may also be present, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers, antioxidants and preservatives.

Methods of administering recombinant virulence-attenuated Gram-negative bacteria to a subject can be selected from the group consisting of intravenous, intratumoral, intraperitoneal, and oral administration. Although the present invention is not intended to be limited to any particular mode of administration, intravenous or intratumoral administration of bacteria or pharmaceutical compositions is preferred.

Depending on the route of administration, the active ingredients that contain bacteria may be required to be coated with a material to protect said organisms from enzymes, acids and other natural conditions that may inactivate said organisms. For the purpose of administering bacteria by a route other than parenteral administration, the bacteria must be coated with a material to protect against inactivation or must be administered together with the specified material. For example, bacteria can be administered together with enzyme inhibitors or in liposomes. Enzyme inhibitors include pancreatic trypsin inhibitor, diisopropyl fluorophosphate (DFP), and trazylol. Liposomes include P40 water-in-oil-in-water emulsions, as well as conventional and specially formulated liposomes that transport bacteria, such as Lactobacillus, or their byproducts, to the internal target of the host subject.

One bacterium can be administered on its own or in combination with a second, different bacterium. Any number of different bacteria may be used in combination. By in combination with is meant together, substantially simultaneously or sequentially. The compositions may also be administered in the form of a tablet, dragee or capsule, such as a freeze-dried capsule containing the bacteria or pharmaceutical compositions of the present invention, or as a frozen solution of the bacteria or pharmaceutical compositions of the present invention containing DMSO or glycerol. Another preferred form of use involves the preparation of lyophilized capsules of bacteria or pharmaceutical compositions according to the present invention. Another preferred form of use involves the preparation of heat-dried capsules of bacteria or pharmaceutical compositions according to the present invention.

The recombinant virulence-attenuated gram-negative bacteria or pharmaceutical composition to be administered can be administered by injection. Forms suitable for injectable use include monoseptic or sterile aqueous solutions (in the case of water-soluble components) or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions immediately before use. In all cases, the form must be monoseptic or sterile and must be fluid to the extent that it can be easily administered by syringe. The shape must be stable under production and storage conditions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyhydric alcohol (eg, glycerol, propylene glycol, liquid polyethylene glycol, etc.), suitable mixtures of these components and vegetable oils. Proper fluidity can be maintained, for example, through the use of a coating such as lecithin, by maintaining the required particle size in the case of a dispersion. In many cases it will be preferable to include isotonic substances, such as sugars or sodium chloride, in the composition. Prolonged absorption of injectable compositions can be achieved by using substances in the composition that delay absorption, for example, aluminum monostearate and gelatin.

In some embodiments of the present invention, the recombinant attenuated

- 45 044058 virulence gram-negative strain of bacteria is administered to the subject together with a siderophore. These embodiments are preferred. Siderophores that can be co-administered are siderophores including hydroxamate, catecholate and mixed ligand siderophores. Preferred siderophores are deferoxamine (also known as desferrioxamine B, desferoxamine B, DFO-B, DFOA, DFB or Desferal), desferrioxamine E, deferasirox (Exjade, Desirox, Defridget, Desifer) and deferiprone (Ferryprox), with deferoxamine being more preferred. Deferoxamine is a bacterial siderophore produced by the actinobacterium Streptomyces pilosus, which is commercially available, for example, from Novartis Pharma Schweiz AG (Switzerland).

Co-administration with a siderophore can be carried out before, simultaneously with or after the introduction of a recombinant virulence-attenuated gram-negative strain of bacteria. Preferably, the siderophore is administered before the introduction of a recombinant virulence-attenuated gram-negative bacterial strain, more preferably administered at least 1 hour before, preferably at least 6 hours before, more preferably at least 12 hours before, in particular at least 24 hours before the subject is administered a recombinant virulence-attenuated gram-negative strain of bacteria. In a specific embodiment, the subject is pretreated with desphreoxamine 24 hours prior to infection with a recombinant virulence-attenuated Gram-negative bacterial strain to promote bacterial growth. Typically, the siderophore is co-administered in a single dose of from about 0.5x10'5 mole to about 1x10'3 mole, more preferably from about 1x10'5 mole to about ^1x10'4 mole, preferably from about 3.5x10'5 mole to about 1.1x10'4 mol per 1 kg of body weight. Typically, desferoxamine is coadministered at a single dose of about 20 mg to about 60 mg, preferably about 20 mg to about 60 mg, per kg of body weight.

Dosage regimens for administering a recombinant virulence-attenuated Gram-negative bacterial strain or pharmaceutical composition described herein will vary depending on the specific purpose intended, the age and physical condition of the subject being treated, the duration of treatment, the nature of the concomitant therapy, and the specific bacterium used. as known to the specialist. The recombinant virulence-attenuated Gram-negative bacterial strain is typically administered to a subject according to a dosing regimen consisting of a single dose every 1-20 days, preferably every 1-10 days, more preferably every 1-7 days. The administration period is usually from about 20 to about 60 days, preferably about 30-40 days. Alternatively, the administration period is typically from about 8 to about 32 weeks, preferably from about 8 to about 24 weeks, more preferably from about 12 to about 16 weeks.

In a further embodiment, the present invention provides a kit for treating cancer, such as, for example, malignant solid tumors, preferably in humans. Such kits will typically contain a recombinant virulence-attenuated Gram-negative bacterial strain or pharmaceutical composition described herein and instructions for use of the kit. In some embodiments, the kits contain a carrier, package, or container that is divided into one or more containers, such as vials, tubes, and the like, each of the container or containers containing one of the individual elements for use in the method described in this document. Suitable containers include, for example, bottles, vials, syringes and laboratory tubes. In other embodiments, the containers are made from a variety of materials, such as glass or plastic.

Examples

Example 1.

A) Materials and methods.

Bacterial strains and growth conditions.

The strains used in this study are listed in Fig. FOR-M. E. coli Top10, which was used for plasmid purification and cloning, and E. coli Sm10λ pir, which was used for conjugation, as well as E. coli BW19610 ³¹ , which was used for propagation of pKNG101, were grown in the conventional manner on LB agar plates and in broth LB at 37°C. Ampicillin was used at a concentration of 200 μg/ml (Yersinia) or 100 μg/ml (E. coli) for selection of expression vectors. Streptomycin was used at a concentration of 100 μg/ml to select suicide vectors. Y. enterocolitica MRS40 (0:9, biotype 2) ²⁰ , an E40 derivative ¹⁹ that is not resistant to ampicillin, and strains obtained from it were grown in a conventional manner on brain heart extract (Brain Heart Infusion BHI; Difco) at RT. (room temperature). Nalidixic acid (35 μg/ml) was added to all Y. enterocolitica strains, and 100 μg/ml meso-2,6-diaminopimelic acid (mDAP, Sigma Aldrich) was additionally added to all Y. enterocolitica asd strains. S. enterica SL1344 was grown conventionally on LB agar plates and in LB broth at 37°C. Ampicillin is used

- 46 044058 was called at a concentration of 100 μg/ml for the selection of expression vectors in S. enterica.

Genetic manipulations of Y. enterocolitica.

Genetic manipulation of Y. enterocolitica has been described ^32,33 . Briefly, mutators to modify or delete genes in pYV plasmids or on the chromosome were constructed by PCR with overlapping 2-fragment primers using purified pYV40 plasmid or genomic DNA as a template, resulting in flanking sequences of 200–250 bp. O. on both sides of the deleted or modified part of the corresponding gene. The resulting fragments were cloned into pKNG101 ²⁹ in E. coli BW19610 ³¹ . E. coli Sm10X pir was transformed with sequence-confirmed plasmids, from which the plasmids were mobilized into the corresponding strain of Y. enterocolitica. Mutants carrying the integrated vector were propagated to produce multiple generations without selection pressure. Sucrose was then used to select clones that had lost the vector. Finally, mutants were identified using colony selection PCR. Specific mutators (pSi_408, pSi_419) are listed in Table. III.

Plasmid construction.

Plasmid pBad_Si2 or pBad_Si1 (Fig. 2) was used to clone fusion proteins with the N-terminal 138 amino acids of YopE (SEQ ID NO: 2). pBad_Si2 was constructed by cloning the SycE-YopE _1-138 fragment-containing endogenous promoters for YopE and SycE from purified pYV40 into the KpnI/HindIII site of pBad-MycHisA (Invitrogen). Additional modifications include removal of the NcoI/BglII fragment of pBad-MycHisA by digestion, treatment with Klenow fragment, and religation. A bidirectional transcription terminator (BBa_B1006; iGEM foundation) was cloned into a KpnI cut treated with a Klenow fragment (pBad_Si2) or a BglII cut site (pBad_Si1). The following cleavage sites were then added at the 3′ end of YopE _1-138 : Xbal-XhoI-BstBI-(HindIII) (Fig. 2 B). pBad_Si1 is similar to pBad_Si2 but encodes EGFP amplified from pEGFP-C1 (Clontech) at the NcoI/BglII site under the control of an arabinose-inducible promoter. PSI_266, psi_267, psi_268 and psi_269 plasmids containing the corresponding endogenous promoter and fragment Stea _1-20 (PSI_266), a full-size sequence of Stea (PSI_267), fragment Sope _1-81 (PSI_268) or fragment SOPE _1-105 (PS I_269), amplified from genomic DNA of S. enterica SL1344 and cloned into the NcoI/KpnI site of pBad-MycHisA (Invitrogen).

Full-length genes or their fragments were amplified with specific primers listed in Table. I below and cloned as fusions with YopE _1-138 into plasmid pBad_Si2 or, in the case of z-BIM (SEQ ID NO: 16), into pBad_Si1 (see Table II below). For fusion with SteA or SopE, synthetic DNA constructs were digested with KpnI/HindII and cloned into pSi_266, pSi_267, pSi_268, or pSi_269, respectively. In the case of genes from bacterial species, purified genomic DNA was used as a template (S. flexneri M90T, Salmonella enterica subspecies enterica serovar Typhimurium SL1344, Bartonella henselae ATCC 49882). For human genes, a universal DNA library (Clontech) was used unless otherwise noted (Fig. 3A-M, zebrafish genes were amplified from a DNA library (kindly provided by M. Affolter)). The ligated plasmids were cloned into E. coli Top10. Sequenced plasmids were transferred by electroporation into the desired strain of Y. enterocolitica or S. enterica using the settings for standard E. coli electroporation.

Table I No. Si Primer_: Sequence

Seq_Id_No_51: Primer No.: Si_285

CATACCATGGGAGTGAGCAAGGGCGAG

Seq_Id_No_52: Primer No.: Si_286

GGAAGATCTttACTTGTACAGCTCGTCCAT

Seq_Id_No_53: Primer No.: Si_287

CGGGGTACCTCAACTAAATGACCGTGGTG

Seq_Id_No_54: Primer No.: Si_288

GTT AA AGC TTttcgaatctagactcgagCGT GGCGA ACTGGTC

Seq_Id_No_55: Primer No.: Si_387

CGTAtctagaATGGACTGTGAGGTCAACAA

Seq_Id_No_56: Primer No.: Si_391 CGTAtctagaGGCAACCGCAGCA

Seq_Id_No_57: Primer No.: Si_389

GTTAAAGCTTTCAGTCCATCCCATTTCTg

Seq_Id_No_58: Primer No.: Si_436

CGTAtctagaATGCCCCGCCCC

- 47 044058

Seq_Id_No_59: Primer No.: Si_437

GTTAAAGCTTCTACCCACCGTACTCGTCAAT

Seq_Id_No_60: Primer No.: Si_438

CGTAtctagaATGTCTGACACGTCCAGAGAG

Seq_Id_No_61: Primer No.: Si_439

GTTAAAGCTTTCATCTTCTTCGCAGGAAAAAG

Seq_Id_No_62: Primer No.: Si 463

C AGT ctcgaggaaagcttgtttaaggggc

Seq_Id_No_63: Primer No.: Si_464 cagtTTCGAAttagcgacggcgacg

Seq_Id_No_64: Primer No.: Si_476

GTTAAAGCTTttACTTGTACAGCTCGTCCAT

Seq_Id_No_65: Primer No.: Si_494

CGTAtctagaATGGCCGAGCCTTG

Seq_Id_No_66: Primer No.: Si_495

GTTAAAGCTTttaTTGAAGATTTGTGGCTCC

Seq_Id_No_67: Primer No.: Si_504

CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTTCAAAGTA TGCCCCGCCCC

Seq_Id_No_68: Primer No.: Si_505

GTTAAAGCTTCCCACGTACTCGTCAATtc

Seq_Id_No_69: Primer No.: Si_508

CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTTCAAAGTA TGGCCGAGCCTTG

Seq_Id_No_70: Primer No.: Si_509

GTTAAAGCTTTTGAAGATTTGTGGCTCCc

Seq_Id_No_71: Primer No.: Si_511

CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTTCAAAGTG TGAGCAAGGGCGAG

Seq_Id_No_72: Primer No.: Si_512

CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTTCAAAGTC CGCCGAAAAAAAAACGTAAAGTTGTGAGCAAGGGCGAG

Seq_Id_No_73: Primer No.: Si_513

GTTAAAGCTTttAAACTTTACGTTTTTTTTTCGGCGGCTTGTACAGCTCGTCC AT

- 48 044058

Seq_Id_No_74: Primer No.: Si_515

CGTAtctagaGAAAATCTGTATTTTCAAAGTGAAAATCTGTATTTTTCAAAGTG ATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG

Seq_Id_No_75: Primer No.: Si_677

TTACTATTCGAAGAAATTATTCATAATATTGCCCGCCATCTGGCCCAAATT GGTGATGAAATGGATCATTAAGCTTGGAGTA

Seq_Id_No_76: Primer No.: Si_678

TACTCCAAGCTTAATGATCCATTTCATCACCAATTTGGGCCAGATGGCGGG

CAATATTATGAATAATTTCTTCGAATAGTAA

Seq_Id_No_77: Primer No.: Si_682

TTACTACTCGAGAAAAAACTGAGCGAATGTCTGCGCCGCATTGGTGATGA ACTGGATAGCTAAGCTTGGAGTA

Seq_Id_No_78: Primer No.: Si_683

TACTCCAAGCTTAGCTATCCAGTTCATCACCAATGCGGCGCAGACATTCGC TCAGTTTTTTCTCGAGTAGTAA

Seq_Id_No_79: Primer No.: Si_580 catgccatggatttatggtcatagatatgacctc

Seq_Id_No_80: Primer No.: Si_612

CGGGGTACCatgaggtagcttatttcctgataaag

Seq_Id_No_81: Primer No.: Si_613

CGGGGTACCataattgtccaaatagttatggtagc

Seq_Id_No_82: Primer No.: Si_614 catgccatggCGGCAAGGCTCCTC

Seq_Id_No_83: Primer No.: Si_615 cggggtaccTTTATTTGTCAACACTGCCC

Seq_Id_No_84: Primer No.: Si_616 cggggtaccTGCGGGGTCTTTTACTCG

Seq_Id_No_85: Primer No.: Si_585

CAGTctcgagATGCAGATCTTCGTCAAGAC

Seq_Id_No_86: Primer No.: Si_586

GTTAAAGCTTgctagcttcgaaACCACCACGTAGACGTAAGAC

Seq_Id_No_87: Primer No.: Si_588 cagtTTCGAAGATTATAAAGATGATGATGATAAAATGGCCGAGCCTTG

Seq_Id_No_88: primer no. 733

TTACTACTCGAGGGTGCCATCGATGCCGAAGAAATTATTCATAATATTGCC CG

- 49 044058

Seq_Id_No_89: Primer No. 735

TACTCCTTTCGAATTAATGATCCATTTCATCACCAATTTG

Seq_Id_No_90: primer no. 736

TTACTACTCGAGGGTGCCATCGATGCCAAAAACTGAGCGAATGTCTGCG

Seq_Id_No_91: primer no. 738

TACTCCTTCGAATTAGCTATCCAGTTCATCACCAATG

Seq_Id_No_92: Primer No. 734

TACTCCTTCGAAGGCACCATGATCCATTTCATCACCAATTTGG

Seq_ID_No_93: Primer No. 725:

TTACTATTCGAAGAAATTATTCATAATATTGCC

Seq_ID_No_94: Primer No. 726:

TACTCCAAGCTTACGGTTGAATATTATGATCCATTTCATCACCAATTTGG

Seq_ID_No_95: Primer No. 727:

TTACTATTCGAAGCCGGTGGTGCCGAAGAAATTATTCATAATATTGCCC

Seq_ID_No_96: Primer No. 728:

TACTCCAAGCTTAATGATCCATTTCATCA

Seq_ID_No_97: Primer No. 737:

TACTCCTTCGAAGGCACCGCTATCCAGTTCATCACCAATG

Seq_ID_No_101: Primer No. 869:

gatcgtcgacTTAAGTTCAATGGAGCGTTTAATATC

Seq_ID_No_102: Primer No. 870:

ctttgactggcgagaaacgcT C TT A AC AT GAGGC T GAGC T C

Seq_ID_No_103: Primer No. 871:

GAGCTCAGCCTCATGTTAAGAgcgtttctcgccagtcaaag

Seq_ID_No_104: Primer No. 872:

gatagccccgagcctgtGCACTTTGTCATTAACCTCAGC

Seq_ID_No_105: Primer No. 873:

GCTG AGGTT A AT GAC A A AGT GCacaggctcggggggctatc

Seq_ID_No_106: Primer No. 874:

catgtctagaCCCTCAGCATAATAACGACTC

Seq_ID_No_107: Primer No. 600:

catgacatgtTGGCGTTTCTCGCC

Seq_ID_No_108: Primer No. 601:

- 50 044058 catgacatgtATTAACCTCAGCCCTGACTATAAG

Seq_ID_No_l 19: primer no. 1010:

cacatgtctagaCAACCGTTTCCGAAAGGTGATCTG

Seq_ID_No_l20: primer no. 1012: atccCAagctTATTGGCGTTGGGTGGTAAAAATTTTG

Seq_ID_No_l2l: primer no. 1021: cacatgtctagaATGACCGCCGAACAACGC

Seq_ID_No_l22: primer no. 1022: catgaagcttaCGGACCCGGATTTTGGCTC >Seq_ID_No_123: primer no. 1023:

catgaagcttaCGGTTCTTCTTGAATAAAAATTTGAATG

Seq_ID_No_l24: primer no. 1024: catgaagcttaTTGCAGCACTTTCGGCCAATTT

Seq_ID_No_l25: primer no. 1025:

cacatgtctaga AT GAGC ATTGT GT GT AGC GC

Seq_ID_No_l26: primer no. 1026: catgaagcttaGCTTTCATCCACGGCCGG

Seq_ID_No_l27: primer no. 1027: catgaagcttaATTACCGGTTTGGCGCAGC

Table II Cloned fusion proteins

Protein that will be delivered by T3SS Seq. ID. Protein No. Framework plasmid Name of the resulting plasmid Primers. Si_Nr.: Seq. Primer ID no. YopEl-138-MycHis 3 pBadMycHisA (Invitrogen) pBadSil 285/286 (EGFP), 287/288 (sycEYopEl138) 51/52 and 53/54 Y orE 1-138 -Mu cHi s 3 pBadMycHisA (Invitrogen) pBad_Si_2 287/288 (sycE- 53/54

- 51 044058

YopEl138) YopEl-138- Bid person 16 pBad_Si_2 pSi_85 387/391 55/56 YopEl-138-t-Bid person 17 pBad_Si_2 pSi_87 389/391 55/57 YopEl-138-ETl 9 pBad_Si_2 pSi_120 436/437 58/59 YopEl-138-z-BIM 16 pBadSil pSi_121 438/439 60/61 YopEl-138 protease TEV S219V 12 pBad_Si_2 pSi_132 463/464 62/63 YopEl-138-Ink4C 8 pBad Si 2 pSi 151 494/495 65/66 YopEl-138-2x website TEV-ET1 AND pBad_Si_2 pSi_156 504/505 67/68 YopEl-138-2x TEV-EGFP site 98 pBad_Si_2 pSi_158 511/476 71/64 YopEl-138-2x TEV site - EGFP - NLS 99 pBad_Si_2 pSi_159 511/513 71/73 YopEl-138-2x TEV site - NLS - EGFP 100 pBad_Si_2 pSi_160 512/476 72/64 YopEl-138-2x TEV site - INK4C 10 pBad_Si_2 pSi_161 508/509 69/70 YopEl-138-2x website TEV - Flag - INK4C 13 pBad_Si_2 pSi_164 515/509 74/70 Y opE 1-13 8-code oh optimized for Y. enterocolitica part BH3 tBid mouse 19 pBad_Si_2 pSi_318 677/678 75/76 Y opE 1-13 8-code optimized for Υ. enterocolitica part of VNZ Bax mouse 20 pBad_Si_2 pSi_322 682/683 77/78 SteAl-20 5 pBadMycHisA (Invitrogen) pSi_266 580/612 79/80 SteA 4 pBadMycHisA (Invitrogen) pSi_267 580/613 79/81 SopEl-81 6 pBadMycHisA (Invitrogen) pSi_268 614/615 82/83 SopEl-105 7 pBadMycHisA (Invitrogen) pSi_269 614/616 82/84 Y opE 1-13 8-code optimized for Y. enterocolitica tBid mouse 21 pBad_Si_2 pSi_315 synthetic construction /

- 52 044058

YopEl-138- Ubiquitin 14 pBad_Si_2 pSi_236 585/586 85/86 YopEl-138Ubiquitin-Flag- INK4C-MycHis 15 pSi_236 pSi_237_II 588/509 87/70 Y opE 1-138 - (code optimized for Y. enterocolitica part of mouse BH3 tBid), ready for insertion of additional domains 25 pBad_Si_2 pSi_357 733/735 88/89 Y opE 1-138 - (code optimized for Y. enterocolitica part of the mouse BNC BAC), ready for insertion of additional domains 26 pBad_Si_2 pSi_358 736/738 90/91 Y opE 1-13 8-(codon-optimized for Y. enterocolitica part of the MOUSE tBid VNZ)2 27 pSi_357 pSi_371 733/734 88/92 Y opE 1-(13 8-code on-optimized for Y. enterocolitica part of the mouse BNC tBid - codon-optimized for Y. enterocolitica part of the mouse BHC BAC 28 pSi_358 pSi_373 733/734 88/92 YopEl-138 - codon-optimized elongated part of the mouse VNZ tBid 22 pBad_Si_2 pSi_353 725/726 93/94 ΥορΕι-138-linker 10 AK - codon-optimized part of the tBid mouse VNZ for Y. enterocolitica 23 pBad_Si_2 pSi_354 727/728 95/96 ΥθρΕΐ-138-CODON-optimized for Y. enterocolitica part of mouse VNZ Bax-codon- 24 pSi_357 pSi_374 736/737 90/97

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part of mouse VNZ tBid optimized for Y. enterocolitica ΥθρΕΐ-138-codon-optimized for Y. enterocolitica two human RIG-1 CARD domains (AK 1-245) 37 pBad_Si_2 pSi_453 synthetic construction / ΥθρΕΐ-138-codon-optimized for Y. enterocolitica two mouse RIG-1 CARD domains (AK 1246) 38 pBad_Si_2 pSi_454 synthetic construction / YopEl-138- codon-optimized for Y. enterocolitica GCN4 S. cerevisiae (AK 249-278) codon-optimized for Y. enterocolitica WspRP. aeruginosa (AK 172-347) 39 pBad_Si_2 pSi_452 synthetic construction / YopEl-138-codon-optimized for mouse Y. enterocolitica IRF3 S397D 40 pBad_Si_2 pSi_428 synthetic construction / YopEi.i38- codon-optimized for Y. enterocolitica DncV V. Cholerae (M3toL413) 41 pBad_Si_2 pSi_482 synthetic construction / YopEl-138-CODON optimized for Y. enterocolitica Di sA-like protein B. cereus_ (PDB: 2FB5; AK 76205) 42 pBad_Si_2 pSi_483 synthetic construction / YopEl-138- codon-optimized for Y. enterocolitica cGAS anemone (N. vectensis) (Ensembl: A7SFB5.1) 43 pBad_Si_2 pSi_484 synthetic construction / YopEl-138 - codon-optimized 110 pBad_Si_2 pSi_521 1021/1022 122/12 3

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for Y. enterocolitica mouse CARD RIG1 domains (AK 1-229) YopEl-138 - Y. enterocolitica codon-optimized mouse RIG1 CARD domains (AK 1-218) 111 pBad_Si_2 pSi_522 1021/1023 122/12 4 YopEl-138 - codon-optimized for mouse Y. enterocolitica MDA5 (AK 1294) 112 pBad_Si_2 pSi_517 synthetic construction / YopEl-138 - codon-optimized for mouse Y. enterocolitica MDA5 (AK 1231) 113 pBad_Si_2 pSi_524 1025/1026 126/12 7 YopEl-138- codon-optimized for human Y. enterocolitica cGAS (AK 161-522) 115 pBad_Si_2 pSi_515 synthetic construction / YopEl-138- codon-optimized for human Y. enterocolitica CARD MAVS (AK 1-100) 116 pBad_Si_2 pSi_539 synthetic construction / YopEl-138- codon-optimized for Y. enterocolitica cGAS anemone (N. vectensis) (AK 60422) (Ensembl: A7SFB5.1) 117 pBad_Si_2 pSi_503 1010/1012 120/12 1 YopEl-138- codon-optimized for Y. enterocolitica CdaA Listeria (AK 101-273) 118 pBad_Si_2 pSi_518 synthetic construction /

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Table III

Mutators for genetic modification

Mutator/construction To be inserted into: Framework plasmid The name of the result Primers, Si_Nr.: Seq.Id No. of primers Applied with specific those plasmids give birth lski m strain ohm YopEl-138VNZ tBID mouse pYV pKNGlOl pSi_408 Synthetic gene / / YopEl-138(VNZ tBID MOUSE)2 pYV pKNGlOl pSi_437 Synthetic gene / Strain 5 mutated pSi_40 8 YopEl-138 codon optimized for Y. enterocolitica two mouse CARD RIG-1 domains (AK 1246) pYV pKNGlOl pSi_456 (Seq ID No. 50) Synthetic gene / / pYV-asd pYV pKNGlOl pSi_417 PCR1: 869/870; PCR2: 871/872; PCRZ: 873/874; PCR from overlap PCR1: 101/102; PCR2: 103/104; PCRZ: 105/106; PCR from overlap Nasd

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using ISI primers 869/874 using ISI primers 101/106 pYV-virFhairpinl pYV pKNGlOl pSi_441 Synthetic gene / / pYVpAra-VirF pYV pKNGlOl pSi_439 Synthetic gene / /

Secretion of Yop.

Induction of the yop regulon was carried out by transferring the culture to 37°C in BHI-Ox (permissive conditions for secretion) ³⁴ . Glucose (4 mg/ml) was added as a carbon source. Total cells and supernatant fractions were separated by centrifugation at 20,800 g for 10 min at 4°C. The cell sediment was collected as a total cell fraction. Proteins in the supernatant were precipitated with 10% trichloroacetic acid (w/v) in a final step for 1 h at 4°C. After centrifugation (20,800 g for 15 min) and removal of the supernatant, the resulting precipitate was washed in ice-cold acetone overnight. The samples were centrifuged again, the supernatant was discarded, the pellet was air dried and resuspended in 1x SDS and loading dye.

Secreted proteins were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis); in each case, proteins secreted by 3x108 bacteria were applied to the lane. Detection of specific secreted proteins by immunoblotting was performed using 12.5% SDS-PAGE gels. To detect proteins in total cells, 2x108 bacteria were loaded per lane unless otherwise noted, and proteins were separated on 12.5% SDS-PAGE gels prior to detection by immunoblotting.

Immunoblotting was performed using rat monoclonal antibody against YopE (MIPA193-13A9; 1:1000, ³⁵ ). Antiserum was preabsorbed twice overnight against Y. enterocolitica ΔΗΟΡΕΜΑΤ asd to reduce background staining. Detection was performed using secondary antibodies directed against rat antibodies and horseradish peroxidase-conjugated (1:5000; Southern biotech), followed by imaging with ECL chemiluminescent substrate (LumiGlo, KPM).

Cell culture and infection.

HeLa Ccl2 and B16F10 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% FBS (fetal bovine serum) and 2 mM L-glutamine (cDMEM). 4T1 cells were cultured in RPMI 1640 supplemented with 10% FBS and 2 mM Lglutamine. Y. enterocolitica was grown in supplemented BHI overnight at RT, diluted in fresh BHI to an OD ₆₀₀ of 0.2, and grown for 2 h at RT. until the temperature rises to 37°C on a water bath shaker for another 30 min or 1 h if EGFP is delivered. Finally, bacteria were collected by centrifugation (RCCA, 6000, 30 s) and washed once with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. Finally, bacteria were collected by centrifugation (6000 RCC, 30 s) and washed once with DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. Cells that were seeded in 96-well (for immunofluorescence analysis) or 6-well (for Western blotting) plates were infected at the indicated MOIs in DMEM supplemented with 10 mM HEPES and 2 mM L-glutamine. After adding bacteria, the plates were centrifuged for 1 min at 1750 rpm and placed at 37°C for the indicated periods of time. Extracellular bacteria were killed with gentamicin (100 mg/ml) when indicated. For immunofluorescence assays, infectivity assays were stopped by fixation in 4% PFA (paraformaldehyde). For Western blot analysis, cells were washed twice with ice-cold PBS, and PhosphoSafe lysis buffer (Novagen) was added to lyse the cells. After incubation on ice, the cells were centrifuged (16,000 RCU, 25 min, 4°C).

Supernatants were collected and analyzed for total protein content by the Bradford bicinchoninic acid method (Pierce), followed by SDS-PAGE and Western blotting using anti-actin (Millipore), anti-Bid (Cell Signaling), anti-Myc (Santa) antibodies. Cruz), anti-caspase-3 p17 (Cell Signaling) and anti-Ink4C (Cell Signaling).

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Western blot analysis of T3SS-transposed proteins from infected cells. HeLa cells in 6-well plates were infected at MOI 100 as described above. In the case of coinfection with a TEV protease translocating strain of Y. enterocolitica, an OD of ₆₀₀ strains was set, and the two bacterial suspensions were mixed in a 1:1 ratio in a tube before being added to the cells (unless otherwise stated). At the end of infection, cells were washed twice with ice-cold PBS and collected by scraping into a small volume of ice-cold PBS. After centrifugation (16,000 RCC, 5 min, 4°C), the sediment was dissolved in 0.002% digitonin with the addition of a protease inhibitor cocktail (Roche complete, Roche). The dissolved sediment was incubated for 5 min on ice and then centrifuged (16,000 RCC, 25 min, 4°C). Supernatants were collected and analyzed for total protein content by the Bradford bicinchoninic acid method (Pierce), followed by SDS-PAGE and Western blotting using anti-Myc (Santa Cruz, 9E11) or anti-Ink4C (Cell Signaling) antibodies. .

Automated microscopy and image analysis. Images were automatically acquired using an ImageXpress Micro system (Molecular devices, Sunnyway, USA). Quantitative determination of anti-Myc staining intensities was carried out using MetaXpress software (Molecular devices, Sunnyway, USA). Regions within cells, excluding nuclear regions and regions containing bacteria, were manually selected (40 pixel circles), and the average intensity was recorded.

Biodistribution in B16-F1O and 4T1 tumor allograft mouse models.

All animal experiments were approved (license 1908; Kantonales Veterinaramt BaselStadt) and carried out in accordance with local guidelines (Tierschutz-Verordnung, Basel-Stadt) and the Swiss animal welfare law (Tierschutz-Gesetz). C57Bl/6 and BALB/c mice, 6 weeks old, were ordered from Janvier Labs. After at least one week of adaptation, mice were anesthetized with isoflurane and injected subcutaneously into the flank of C57BP6 and BALB/c with 100 μl of B16P10 or 4T1 cells (1x105-1x106 cells), respectively. Throughout the experiment, the behavior and appearance of the mice, surface temperature, and body weight were measured.

After tumor development, mice were administered Desferal solution 8 mg/ml (10 ml/kg) by i.p. injections. The next day, mice were infected with Y. enterocolitica MRS40 or Y. enterocolitica MRS40 ΔΗΟΡΕΜΑΤ (2x105, 1x106 or ^1x107 bacteria) by tail vein injection. The inoculum, which i.v. administered to mice, confirmed by culture by dilution method. In some experiments, tumor progression was monitored by daily changes in tumor length and width using a digital caliper. Tumor volume was determined as 0.523 x length x width ² . On appropriate days after analysis, mice were sacrificed by CO2 inhalation. A blood sample was immediately collected by cardiac aspiration. The liver, spleen, lungs and tumor were collected and their weight was determined. The organs and tumor were homogenized. CFU in each sample was determined by spot-plating serial dilutions onto LB agar plates containing nalidixic acid (35 μg/ml).

Direct assay for type I interferon activation. B16F10, wild-type mouse RAW264.7 mouse melanoma cells, or MAVS knockout macrophages stably expressing secreted embryonic alkaline phosphatase (SEAP) or secreted Lucia luciferase under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter , enhanced with a multimeric ISRE, was ordered from InvivoGen (B16-Blue ISG, RAW-Blue ISG, RAW-Lucia ISG and RAW-Lucia ISG-KO-MAVS). Growth conditions and type I IFN assays were adapted from protocols provided by InvivoGen. Briefly, 12,500 B16-Blue ISG cells, or 30,000 RAW-Blue, RAW-Lucia, or RAW-Lucia KO-MAVS ISG cells in 150 μl assay medium (RPMI+2mM Lglutamine+10% FBS for B16-Blue ISG cells; DMEM +2mM L-glutamine+10% FBS for RAWBlue, RAW-Lucia and RAW-Lucia KO-MAVS ISG cells) per well were seeded in a 96-well flat bottom plate (NUNC or Corning). The next day, cells were infected with bacterial strains for evaluation by adding 15 μl per well of the desired multiplicity of infection (MOI, diluted in assay medium). After 2 h of incubation (37°C and 5% CO2), bacteria were killed by adding assay medium containing penicillin (100 U/ml) and streptomycin (100 μg/ml). Incubation was continued for 20-24 hours. SEAP and luciferase detection were performed according to QUANTI-Blue™ and QUANTILuc™ protocols (InvivoGen), respectively. For SEAP detection: 20 μl of cell supernatant was incubated with 180 μl of detection reagent (QUANTI-Blue™, InvivoGen). The plate was incubated at 37°C and SEAP activity was measured by reading the OD at 650 nm using a microplate reader (Molecular Devices). Mouse IFN (stock solution: 1,000,000 U/ml) diluted to appropriate concentrations in the assay medium was used as a positive control. For luciferase detection: 50 μL of detection reagent (QUANTI-Luc™, InvivoGen) in opaque plates (ThermoScientific) was added to 20 μl of cell supernatant. Luminescence was measured immediately using a plate reader (BioTek).

Indirect type I interferon activation assay. Mouse B16F10 or 4T1 cells were infected at the indicated multiplicity of infection (MOI) with the bacterial strains assessed for a total of 4 h as described above. The cell supernatant was then transferred to B16F10 mouse melanoma cells stably expressing secreted embryonic alkaline phosphatase (SEAP) under the control of the I-ISG54 promoter (consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE; ordered from InvivoGen, B16-Blue ISG cells). Growth conditions and type I IFN assays were adapted from protocols provided by InvivoGen. Briefly, 12,500 B16-Blue ISG cells in 150 μl assay medium (RPMI+2 mM L-glutamine+10% FBS) per well were seeded in a 96-well flat-bottom plate (NUNC). The next day, all medium was removed and 100 μl of supernatant of cells preinfected with B16F10 or 4T1 was added. The plates were incubated for 2024 hours at 37°C and 5% CO ₂ . SEAP detection was performed according to the QUANTIBlue™ protocol (InvivoGen). 20 μl of cell supernatant was incubated with 180 μl of detection reagent (QUANTI-Blue™, InvivoGen). The plate was incubated at 37°C and SEAP activity was measured by reading the OD at 650 nm using a microplate reader (Molecular Devices).

Study of tumor progression in a B16F10 tumor allograft mouse model after intratumoral treatment. All animal experiments were approved by the responsible authorities and were carried out in accordance with local animal welfare guidelines and laws. Female C57B1/6 mice aged 5-7 weeks were ordered from Charles River (L'Arbresles). After at least one week of adaptation, mice were anesthetized with isoflurane and 1x106 B16-F10 cells in 200 μl RPMI 1640 were injected subcutaneously into the right flank of the mice. At regular intervals, the behavior and appearance of the mice were assessed and body weight was measured.

Treatment began when tumors reached a volume of 60-130 mm ³ (defined as day 0). Mice were administered the bacterial strains assessed on days 0, 1, 2, 3, 6, and 9 by intratumoral injection (7.5 x 107 bacteria in 50 μl PBS per injection) under isoflurane anesthesia. The inoculum that was intratumorally injected into mice was confirmed by dilution culture. As a control, mice were injected with endotoxin-free PBS only. 24 hours before the last administration of bacteria (day 8), mice were administered a solution of Desferal 8 mg/ml (10 ml/kg) by i.p. injections. Tumor progression was monitored by measuring tumor length and width using a digital caliper. Tumor volume was determined as 0.5 x length x width ² . Tumor volume greater than 1500 mm ³ was defined as the end point for humans.

Study of tumor progression and antigenic re-stimulation in EMT-6 mouse tumor allograft models after intratumoral treatment. All animal experiments were approved by the responsible authorities and were carried out in accordance with local animal welfare guidelines and laws. Female BALB/c mice aged 5–7 weeks (BALB/cByJ) were ordered from Charles River (L'Arbresles). After at least one week of adaptation, mice were anesthetized with isoflurane and 1 x 106 EMT-6 cells in 200 μl RPMI 1640 were injected subcutaneously into the right flank of the mice. At regular intervals, the behavior and appearance of the mice were assessed and body weight was measured.

Treatment began when tumors reached a volume of 60-130 mm ³ (defined as day 0). Mice were administered the bacterial strains assessed on days 0, 1, 5, 6, 10, and 11 by intratumoral injection (7.5 x 107 bacteria in 50 μl PBS per injection) under isoflurane anesthesia. The inoculum that was intratumorally injected into mice was confirmed by dilution culture. As a control, mice were injected with endotoxin-free PBS only. 24 hours before the last administration of bacteria (day 10), mice were administered a solution of Desferal 8 mg/ml (10 ml/kg) by i.p. injections. Tumor progression was monitored by measuring tumor length and width using a digital caliper. Tumor volume was determined as 0.5 x length x width ² . Tumor volume greater than 1500 mm ³ was defined as the end point for humans. Mice that showed complete tumor regression on day 54 after the start of treatment were anesthetized with isoflurane, and 1x106 EMT-6 cells in 200 μl RPMI 1640 were injected subcutaneously into the opposite (left) side relative to the first tumor cell injection. included mice that had not previously been used in experiments and had not previously been transplanted with EMT6 cells. Tumor progression was monitored by measuring tumor length and width using a digital caliper. Tumor volume was determined as 0.5 x length x width ² . Tumor volume greater than 1500 mm ³ was defined as the end point for humans.

Study of tumor progression in EMT-6 tumor allograft mouse models after intravenous treatment. All animal experiments were approved by the responsible authorities and were carried out in accordance with local animal welfare guidelines and laws. Female BALB/c mice aged 5–6 weeks (BALB/cByJ) were ordered from Charles River (L'Arbresles). After at least one week of adaptation, mice were anesthetized with isoflurane and 1 x 106 EMT-6 cells in 200 μl RPMI 1640 were injected subcutaneously into the right flank of the mice. At regular intervals, the behavior and appearance of the mice were assessed and body weight was measured.

Mice were randomized to treatment groups when tumors reached a volume of 80-250 mm ³ (defined as day 0). 24 hours before randomization (D-1), mice were injected with Desferal solution 8 mg/ml (10 ml/kg)

- 59 044058 by IP injections. On day 0, mice were administered the bacterial strains being assessed by intravenous injection (5 x 10 ⁶ bacteria in 100 μl PBS per injection) under isoflurane anesthesia. The inoculum that was intravenously injected into mice was confirmed by dilution culture. As a control, mice were injected with endotoxin-free PBS only. Tumor progression was monitored by measuring tumor length and width using a digital caliper. Tumor volume was determined as 0.5 x length x width ² . Tumor volume greater than 1500 mm3 was defined as the human end point.

Measurement of IFNβ secretion following infection of tumor cell isolates.

All animal experiments were approved (license 1908; Kantonales Veterinaramt BaselStadt) and carried out in accordance with local guidelines (Tierschutz-Verordnung, Basel-Stadt) and the Swiss animal welfare law (Tierschutz-Gesetz). 6-week-old BALB/c mice were ordered from Janvier Labs. After a week of adaptation, mice were anesthetized with isoflurane, and 100 μl of EMT-6 cells (1 x 10 ⁶ cells) were injected subcutaneously into the flank of the mice. Throughout the experiment, the mice's behavior and appearance, surface temperature, and body weight were measured. Tumor progression was monitored by measuring tumor length and width using a digital caliper. Tumor volume was determined as 0.5 x length x width ² . On the day of analysis, tumors were collected, cut into small pieces of 1–2 mm, digested for 1–1.5 hours, and passed through a 70-μm nylon mesh to obtain a single-cell suspension. The cell number of a given crude cell isolate was determined and 300,000 cells per well were seeded into a 24-well flat-bottom plate (Corning) in growth medium (DMEM+L-glutamine+nonessential amino acids+10% FBS). After 1 hour of incubation at 37°C and 5% CO _{2 ,} the cells were infected with bacterial strains, which were assessed by adding 100 μl per well of bacterial titer (various MO, dilution in growth medium). After 1 h of incubation (37°C and 5% CO ₂ ), bacteria were killed by adding growth medium containing penicillin (100 U/ml) and streptomycin (100 μg/ml). Incubation was continued for an additional 3 hours. The plate was centrifuged to collect all cells at the bottom of the wells, and the concentration of IFNβ in the supernatant was analyzed using the LumiKine™ Xpress mouse IFNβ ELISA (Invivogen) according to the manufacturer's instructions.

B) Results.

Protein delivery system based on the secretion of type 3 YopE fusion proteins.

Although the very N terminus of the Y. enterocolitica T3SS effector YopE (SEQ ID no. 1) contains a secretion signal sufficient for the translocation of heterologous proteins ²² , it does not contain a chaperone-binding site (CBS) for its chaperone ( SycE) ³⁶ . We selected the N-terminal 138 amino acids of YopE (SEQ ID NO: 2) to be fused to the proteins to be delivered because this approach has been shown to perform best in the translocation of other heterologous T3S substrates ²⁴ . Since the N-terminal 138 amino acids of YopE contain CBS, we then decided to coexpress SycE. The SycE-YopE fragment ₁ - ₁₃₈ , cloned from the purified Y. enterocolitica virulence plasmid pYV40, contains the endogenous promoters of YopE and its chaperone SycE (Fig. 2). As a consequence, SycE and any YopE1_ ₁₃₈ fusion protein are induced by a rapid temperature shift from growth at RT. up to 37°C. The culture time at 37°C will influence the amount of fusion protein present in the bacteria. A multiple cloning site (MCS) was added at the 3' end of YopE1_ ₁₃₈ (Fig. 2 B), followed by Myc and a 6x His tag, as well as a stop codon.

The background strain was carefully selected. First, to limit the translocation of endogenous effectors, we used a Y. enterocolitica strain in which all known effectors, Yop H, O, P, E, M, and T, were deleted (termed AHOPEMAT) ³⁷ . In addition, the present inventors have sometimes used an auxotrophic mutant that cannot grow in the absence of exogenous meso-2,6-diaminopimelic acid ³⁸ . The aspartate beta-semialdehyde dehydrogenase (Aasd) gene was deleted in this strain and was classified as biosafety level 1 according to the Swiss Safety Agency (amendment A010088/2). In addition, we have deleted the adhesion proteins YadA and/or InvA to provide greater selection of background strains. While administration of yadA or yadA/invA strains reduced induced background signaling ³⁹ , there was also an effect on the amount of protein delivered ⁴⁰ .

Removal of the YopE ₁ - ₁₃₈ additive after translocation of the fusion protein into a eukaryotic cell.

Despite the fact that the fragment YopE1. ₁₃₈ provides a major advantage for bacterial delivery, it can interfere with the function and/or localization of fusion proteins. As a result, its removal after delivery of proteins will be optimal. To this end, we introduced two TEV cleavage sites (ENLYFQS) ^41,43 between YopE1.138 and the fusion partner (transcription regulator ET1-Myc (SEQ ID NO: 9 and 11) ⁴⁴ and human INK4C (SEQ ID NO: 8 and SEQ ID NO: 10)). To maintain the advantages of the present method, the present inventors further fused the TEV protease (variant S219V; ⁴⁵ ) to _YopE1-138 (SEQ ID NO: 12) in another Y. enterocolitica strain.

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HeLa cells were infected with both strains simultaneously. To ensure analysis of only the translocated fraction of proteins, infected HeLa cells were lysed within 2 h after infection with digitonin, which is not known to lyse bacteria ( ⁴⁶ ;). Western blot analysis revealed the presence of YopE1_138-2x TEV-ET1-Myc cleavage site or YopE1_138-2x TEVFlag-INK4C-Myc cleavage site only when cells were infected with the corresponding strain. After digestion of this cell lysate with purified TEV protease overnight, a shifted band was observed. This band corresponds to ET1-Myc or Flag-INK4C with N-terminal TEV cleavage site residues, most likely serine only. After coinfection of cells with a TEV protease-delivering strain, the same cleaved fragment of ET1-Myc or Flag-INK4C becomes visible; this indicates that the TEV protease delivered by the T3SS is functional and that single cells were infected with both strains of bacteria. Although cleavage is not yet complete, most translocated proteins are cleaved within 2 h postinfection, and even overnight digestion with purified TEV protease did not achieve a higher degree of cleavage. It has been reported that TEV protease‐dependent cleavage may require optimization depending on the fusion protein ^{47 , 48} . Consequently, TEV protease-dependent removal of the YopE1_ ₁₃₈ additive after translocation enables protein delivery via T3SS for the first time for essentially native heterologous proteins with a change in amino acid composition of only one N-terminal amino acid. An alternative approach to TEV protease-dependent cleavage of the YopE fragment is to incorporate ubiquitin into the fusion protein of interest. Indeed, ubiquitin is processed at the C-terminus by a group of endogenous ubiquitin-specific C-terminal proteases (deubiquitinating enzymes, DUBs). Since cleavage is believed to occur at the very C-terminus of ubiquitin (after G76), the protein of interest should not contain any additional amino acid sequence. This method was tested on the YopE ₁ fusion protein. ₁₃₈ -Ubiquitin-Flag-INK4C-MycHis. In control cells infected with bacteria expressing YopE ₁ _ ₁₃₈ -Flag-INK4C-MycHis, a band corresponding to YopE ₁ _ ₁₃₈ -Flag-INK4C-MycHis was detected; this indicates efficient translocation of the fusion protein. When cells were infected for 1 h with YopE expressing ₁ . ₁₃₈ -Ubiquitin-Flag-INK4C-MycHis bacteria, an additional band corresponding in size to Flag-INK4C-MycHis was visualized; this indicates that part of the fusion protein has been cleaved off. This _result demonstrates that the introduction of ubiquitin into the fusion protein allows cleavage of the YopE _{1_138} fragment without the need for an exogenous protease.

Virulence attenuation by deletion/mutation of bacterial effector proteins with virulence activity against eukaryotic cells.

In the case of Y. enterocolitica, virulence was reduced by deletion of six endogenous effector proteins called Yersinia outer proteins (Yop), more specifically YopH, O, P, E, M, T (MRS40 pIML421 [yopHA1-352, yopOA65 -558, yopP23, yopE21, yopM23, yopT135]) ³⁷ . The Yop data are encoded on the Yersinia virulence plasmid (pYV), an approximately 70-kb plasmid that encodes the complete type 3 secretion system (T3SS) as well as other virulence proteins (Fig. 4). YopH, O, P, E, M, and T are six effector proteins that are delivered to host cells by the bacterial type 3 secretion system to modulate and attenuate the immune system. Each Yop has specific biochemical activity in the host cell. YopT cleaves off the C-terminal cysteine of Rho GTPases and thus removes the isoprenyl group that anchors the GTPases to the membrane. This inactivation of Rho due to repositioning allows it to avoid phagocytosis by immune cells such as macrophages and neutrophils ⁴⁹ . In the same pathway, YopE acts as a GTPase activating protein (GAP) for Rho GTPases, deactivating them. This results in decreased levels of phagocytosis and inhibition of immune cell release of IL‐1 beta ⁴⁹ . Moreover, YopO acts as a guanidine nucleotide dissociation inhibitor (GDI), deactivating Rho GTPases. YopO also contains a serine/threonine kinase domain that acts on the actin cytoskeleton in a manner that is not fully understood ⁴⁹ . YopH is a tyrosine phosphatase that acts on focal adhesion proteins such as focal adhesion kinase (Fak), paxillin and others, thereby highly preventing phagocytosis by macrophages and neutrophils ⁴⁹ . YopP, designated YopJ in Y. pseudotuberculosis or Y. pestis, was found to inactivate the MAPK/NFkB pathway in immune cells, preventing the release of TNF-α and IL-8 stimulated by the presence of bacteria from immune cells. Moreover, YopP was found to induce apoptosis in immune cells, which may be due to its effect on the MAPK pathway, which in its activated state protects cells from apoptosis 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). It appears that YopM may stimulate RSK1 phosphorylation and thus affect downstream targets such as, for example, cell cycle progression ⁴⁹ . As a result of deletion

- 61 044058 one or more of the data Yop the protective mechanism of bacteria against the immune system is fundamentally impaired ⁵⁰ . The mutation of the corresponding yops was confirmed by PCR in the corresponding region and by in vitro secretion assay. In vitro secretion analysis by SDS-PAGE and Coomassie blue staining confirmed the absence of full-length YopH,O,M and YopE.

Moreover, a Y. enterocolitica strain with deletions in asd (aspartate semialdehyde dehydrogenase) was constructed. Mutation in asd results in complete loss of growth ability without the addition of mesodiaminopimelic acid. This approach produces antibiotic-free plasmid-maintaining systems based on the presence of asd on the corresponding plasmid. Likewise, other auxotrophic mutants can be used.

Obtaining enhanced proapoptotic bacteria.

To optimize delivery or proapoptotic proteins, strains transformed with various proapoptotic proteins were obtained, according to Table. IV.

Table IV

Strains transformed with various proapoptotic proteins

Strain name Background strain Protein that will be delivered by T3SS Frame I am a plasmid Name of the resulting plasmid Primers. Si_Nr.: Sustainability YopEl-138 (codon optimized for Y enterocolitica elongated part of mouse tBid VNZ) Y enterocolitic a ΔγορΗ,Ο,Ρ, Ε,Μ,Τ Aasd YopEl-138 codon optimized for Y enterocolitic a extended VNZ tBid mouse (by 4 AA) pBad_Si_ 2 pSi_353 Nai Amp YopEl-138linker 10 AK-(codon optimized for Y enterocolitica part of mouse tBid BNZ) Y enterocolitic a ΔγορΗ,Ο,Ρ, Ε,Μ,Τ Aasd YopEl-138 linker 10 AK codon optimized for Y enterocolitic a BNZ tBid mouse pBad_Si_ 2 pSi_354 727/728 Nai Amp YopE 1-(13 8codon optimized for Y Υ enterocolitic a ΔγορΗ,Ο,Ρ, Ε,Μ,Τ Aasd YopEl-138 codon optimized for Y pSi_357 pSi_374 736/737 Nai Amp

- 62 044058

enterocolitica part of VNZ Bax mouse codedon optimized for Y enterocolitica part of VNZ tBid mouse enterocolitic a VNZ Bax mouse codon optimized for Y enterocolitic a VNZ tBID mouse

Shortening the delivered proteins to essential domains required for signaling (eg, the BH3 domain of t-BID (SEQ ID NO: 19)) can increase the efficiency of cell killing (Fig. 5). Without being bound by any theory, it is believed that this increase in efficacy is likely due to an increase in the amount of protein production and subsequent delivery by the T3SS due to the smaller size of the protein delivered. Introducing a linker between the YopE portion and the BH3 domain of tBID (SEQ ID NO: 23) reduced efficiency, as did extending the BH3 domain by 4 additional amino acids (SEQ ID NO: 22) (Fig. 5).

Additionally, synthetic cargoes with repeats of such essential domains (e.g., the BH3 t-BID domain (SEQ ID NO: 27)) or combinations of these essential domains (for example, the BH3 t-BID domain and the BH3 BAX domain (SEQ ID NO: 24 and 28)). Surprisingly, tandem repeats of the same or different BH3 domains were found to result in enhanced induction of apoptosis in cancer cell lines (including 4T1 and B16F10 cells, FIG. 5). The IC ₅₀ (half maximum inhibitory concentration), indicating the number of bacteria per eukaryotic cell (MC) required to kill 50% of eukaryotic cells, was found to be reduced following delivery of BH3 tBID tandem repeats compared to a single BH3 tBID domain (Figure 5). ). This result was unexpected because the size of the protein increases when fused to the second BH3 domain of t-BID. In this regard, one would expect a decrease in the levels of expression and delivery of YopE _1-138 -(BH3 tBID)2 (SEQ ID NO: 27) compared to YopE _1-138 -BH3 tBID (SEQ ID NO: 19 and 25), and at best, equivalent levels could be expected. To achieve enhanced cell killing activity, the BH3 tBID fusion domains must simultaneously act together upon delivery into eukaryotic cells by the T3SS. In the case of only one BH3 tBID domain in YopE _1-138 -(BH3 tBID)2, the design will be functional and, at best, the same efficiency as for YopE _1-138 -BH3 tBID can be expected.

In order to increase the genetic stability of YopE _1-138 -(BH3 tBID)2 (SEQ ID NO: 27) for in vivo studies, the present inventors cloned YopE _1-138 -(BH3 tBID) ₂ (SEQ ID NO: 27) by homologous recombination on the Yersinia pYV virulence plasmid in the native YopE site and under the control of the native YopE promoter (using mutator plasmids pSI_408 npSI_419). Such mutators contain a DNA sequence encoding the desired protein, flanked on both sides by 200-250 bp sequences that correspond to the site of the corresponding gene where integration should take place. These plasmids were used to transform E. coli Sm10λ pir, from which the plasmids were mobilized into the corresponding strain of Y. enterocolitica. Mutants carrying the vector were propagated to produce several generations without selection pressure. Sucrose was then used to select clones that had lost the vector. Finally, mutants were identified using PNR for colony selection. Endogenous proteins for transport by the T3SS (termed Yersinia extrinsic proteins, Yop) are encoded by Y. enterocolitica on a given 70-kb plasmid, called the Yersinia virulence plasmid (pYV), which additionally encodes the T3SS machinery. The ability of Yersinia strains encoding YopE _1-138 -(BH3 tBID) (SEQ ID NO: 19 and 25) or YopE _1-138 (BH3 tBID)2 (SEQ ID NO: 27) on the Yersinia virulence plasmid pYV at the native site of YopE was assessed and under the control of the native YopE promoter, induce apoptosis in cancer cells (including 4T1 and B16F10 cells, Fig. 6). It was found that the IC ₅₀ (half maximum inhibitory concentration), indicating the number of bacteria per eukaryotic cell (MC) required to kill 50% of such cells, is reduced after delivery of tandem repeats of the BH3 tBID domain compared to a single BH3 tBID domain when both proteins are encoded on the Yersinia virulence plasmid pYV at the native YopE site and under the control of the native YopE promoter (Fig. 6). This is consistent with the results obtained with the expression plasmid delivering these proteins (Fig. 5). This result was again unexpected, since the size of the protein increases when fused with the second domain of BH3 t-BID. In this regard, one would expect a decrease in the levels of expression and delivery of YopE1-138-(BH3

- 63 044058 tBID)2 (SEQ ID NO: 27) compared to YopE1. ₁₃₈ -BH3 tBID (SEQ ID NOs: 19 and 25), and at best equivalent levels would be expected. To achieve enhanced cell killing activity, the BH3 tBID fusion domains must simultaneously act together upon delivery by the T3SS into eukaryotic cells. In the case of only one BH3 tBID domain in YopE _1-138 -(BH3 tBID) ₂ the design will be functional and, at best, the same efficiency as for YopE _1-138 -BH3 tBID can be expected. Moreover, the ability of Yersinia strains encoding _YopE1-138 (BH3 tBID) ₂ (SEQ ID NO: 27), on the Yersinia pYV virulence plasmid at the native YopE site and under the control of the native YopE promoter, to induce apoptosis in cancer cells delivered by YopE ₁ was compared _-138 (BH3 tBID) ₂ based on expression plasmid (based on pBad-MycHisA). Consistent with the higher copy number of pBad-MycHisA (20-25 copies) compared to pYV (1-6 copies reported), delivery of YopE _1-138 -(BH3 tBID) ₂ (SEQ ID NO: 27) based on pBad- MycHisA resulted in a slight decrease in the IC ₅₀ value on 4T1 and B16F10 cells (Fig. 6).

Biodistribution studies in a mouse model of melanoma.

To confirm the feasibility of using Gram-negative bacteria with a mutation or mutations in key virulence determinants, such as T3SS effectors, as a tumor-specific carrier, tumor allograft studies were performed in mice using the well-established B16F10 melanoma model (ATCC No. CRL-6475). When p.c. tumors reached a certain size (approximately 100-200 mm ³ ), mice i.v. infected with 2x105 CFU of Y. enterocolitica subspecies palearctica MRS40 or Y. enterocolitica subspecies palearctica MRS40 AuopH,O,P,E,M,T. To ensure bacterial growth, mice were pretreated with desphreoxamine 24 h before infection. Mice infected with the Y. enterocolitica subspecies palearctica MRS40 WT strain had increased scores on measures of appearance and behavior (Fig. 47-48), and they experienced significant weight loss during the first 48 hours of infection (Fig. 49), which forced the authors of the present invention to kill all mice in this group already on day 2 after infection. In contrast, mice infected with the virulence-attenuated strain of Y. enterocolitica subsp. palearctica MRS40 AuopH,O,P,E,M,T did not experience significant weight loss and still scored normal on appearance and behavior assessments ( Fig. 47-49) on day 4 after infection. In mice infected with the WT strain (Y. enterocolitica subspecies palearctica MRS40), live bacteria were found in all organs studied and, moreover, in the blood (Fig. 51). Although DT bacteria were found in the malignant solid tumor, equal or greater numbers of bacteria were found in other organs, most notably in the spleen (Fig. 51). In contrast, in mice infected with Y. enterocolitica subsp. palearctica MRS40 AuopH,O,P,E,M,T, on day 1 postinfection, live bacteria were mainly found in the malignant solid tumor, and the number of bacteria found in the spleen liver and lungs was low. It is noteworthy that on day 4 after infection, the number of bacteria in a malignant solid tumor increased several tens of times (and reached more than 10 ⁸ CFU/g of tumor tissue), while in all other organs studied the number of bacteria decreased below the detection limit (Fig. 50). Thus, Y. enterocolitica subspecies palearctica MRS40 AuopH,O,P,E,M,T accumulated at day 4 postinfection at the site of a malignant solid tumor to a dose approximately (minimum) one million times greater than the dose in the spleen or liver (at calculation of dose versus detection limit).

Similarly high numbers of bacteria per gram of solid tumors on day 5 or 8 after intravenous injection into the lateral tail vein of mice with B16F10 melanoma were determined for Y. enterocolitica dHOPEMT, Y. enterocolitica dHOPEMT+pYV- _YopE1-138 - CARD RIG1 mouse, or Y. enterocolitica dHOPEMT AHairpinI-VirF+pYV-YopE1. ₁₃₈ - CARD RIG1 mouse (Fig. 26). This confirms that heterologous protein cargo and additional mutations affecting T3SS regulation (VirF) do not affect bacterial colonization of solid tumors in mouse models.

Our results support this virulence attenuation strategy by mutating key virulence determinants to produce a bacterial host that specifically targets solid tumor malignancies.

Confirmation of tumor-specific growth in vivo up to day 14 after bacterial administration.

The tumor colonization experiment with genetically modified Y. enterocolitica was repeated in a syngeneic allograft mouse model (4T1 breast cancer model), and bacterial colonization was monitored over a two-week period. This time, mice were infected with 1x106 colony-forming units (CFU) of Y. enterocolitica AyopH,O,P,E,M,T. Although results similar to those obtained in the B16F10 model were obtained during the first days postinfection, we were able to further demonstrate that tumor colonization was consistently detected at day 8 and up to day 14 postinfection (Fig. 7). Moreover, colonization remained highly specific, and only minor numbers of bacteria were detected in all other organs examined (Fig. 8). These results indicate that Y. enterocolitica AyopH,O,P,E,M,T is able to establish persistent colonization of the tumor, thereby preventing clearance by the immune system.

- 64 044058

Efficacy of Y. enterocolitica AHOPEMAT in delaying tumor progression.

To evaluate the effect of YopE _1-138 -(BH3 tBID)2 delivered to tumor cells in vivo, we conducted a study in wild-type Balb/C mice with s.c. allograft of 4T1 breast cancer cells. The aim of the present invention was to evaluate the Y. enterocolitica strain AHOPEMAT, encoding YopE _1-138 -(BH3 tBID)2, on the Yersinia virulence plasmid pYV at the native YopE site and under the control of the native YopE promoter, which was further optimized by deleting the hairpin I region in the direction 3'-5' of VirF to increase the amount of delivered proteins. Mice i.v. were injected with PBS or 1x10 ⁷ Y. enterocolitica AHOPEMAT AHairpinI-virF pYV-YopE1-138-(BH3 tBID)2 when the tumor reached a size of 150-250 mm ³ . Day v.v. injection of bacteria was designated as day 0. Tumor volume was measured over the following days (from day 0 to day 9 after i.v. injection of bacteria) using a caliper. Tumor volume was normalized to tumor volume at day 0 to account for any baseline heterogeneity in tumor size. Treatment with Y. enterocolitica AHOPEMAT AHairpinI-VirF pYV-YopE1-138-(BH3 tBID) ₂ demonstrated an effect on tumor volume progression, with statistically significant tumor reduction at days 8, 9, and 10 after bacteria administration (Figure 9). Importantly, Y. enterocolitica AHOPEMAT alone was found to have no effect on tumor progression in the 4T1 mouse cancer model (Figure 10). These results highlight that such bacteria and their T3SS can be used to inhibit tumor progression.

Y. enterocolitica AHOPEMAT with a deletion in the temperature-sensitive region of RNA in the 3'-5' direction from the gene encoding a DNA-binding protein of the AraC type.

The best known virulence genes of Yersinia are not expressed outside the eukaryotic host and are induced only after entry into the host environment. Expression of these virulence genes is induced by a sharp increase in temperature associated with entry into the host. In this way, pYV-encoded virulence factors such as the T3SS and its effector proteins (Yop) are particularly regulated. At room temperature (20–25°C), the genes required for the regulation of the T3SS, the formation of the T3SS itself, and the delivered effectors are not expressed, and expression is induced only after this increase in temperature to 37°C. Expression of most pYV-encoded virulence genes (yadA and T3SS-associated genes) is temperature-inducible and requires the AraC-type DNA-binding protein VirF in Y. enterocolitica (LcrF in other Yersinia species). Thermoregulation of LcrF expression is thought to occur through the melting at higher temperatures of a loop on the RNA stem into mRNA, which in the unmelted state isolates the ribosome binding site, thereby preventing translation ⁵¹ . In Y. enterocolitica, in contrast, VirF transcription has been shown to depend primarily on temperature ⁵² . More recent studies have demonstrated a more complex picture involving a thermolabile regulator called YmoA ⁵¹ , although a thermosensor RNA 3′–5′ of LcrF has been found to be primarily responsible for temperature regulation of LcrF and thus temperature-dependent genes virulence.

To increase secretion levels of heterologous cargo expressed from pYV under the control of the native YopE promoter, the present inventors aimed to delete one of these RNA hairpin structures in Y. enterocolitica in the 3'-5' direction of VirF. Although the importance of such RNA stem loops in Y. enterocolitica has not been unequivocally demonstrated and temperature induction was more likely to be characteristic of transcriptional changes, we were able to identify hairpin I ⁵¹ 3'-5' of Y. enterocolitica VirF. We then removed by homologous recombination parts of hairpin I (-111 to -57, as in publication ⁵¹ ) and assessed the secretion capacity of the pYV-encoded protein YopE _1-138 -(BH3 tBID) ₂ (SEQ ID NO: 27) or YopE _1-138 - mouse CARD2RIG-1 (SEQ ID NO: 38) (each at the native YopE site) in an in vitro secretion assay (Fig. 11). Surprisingly, it was found that deletion of part of the hairpin I region 3′-5′ of VirF in Y. enterocolitica increased the secretion of heterologous proteins in vitro (Fig. 11 A and B), in contrast to previous reports in Y. enterocolitica ⁵² , in which believed that VirF transcription was the main driving force of thermoregulation.

To allow artificial induction of T3SS expression and protein delivery by T3SS, we replaced the endogenous VirF promoter on pYV with an arabinose-inducible promoter known from pBad-MycHisA in another strain. In addition to replacing the VirF promoter, the present inventors introduced in the 3'-5' direction (in reverse orientation) the complete araC gene. We then assessed the secretion capacity of YopE1-138-(BH3 tBID)2 (SEQ ID NO: 27) (encoded by pYV; at the native site of YopE) in the absence or presence of arabinose in an in vitro secretion assay (FIG. 11A). The heterologous protein was found to be secreted by the T3SS only after the addition of arabinose, consistent with regulation of VirF expression by arabinose.

Y. enterocolitica AHOPEMAT with increased heterologous cargo stability (in vitro and in vivo).

Y. enterocolitia AyopH,O,P,E,M,T has been demonstrated to be highly specific

- 65 044058 strain for targeting solid tumors in the experiments of the authors of the present invention on mice; however, T3SS-dependent delivery of cytotoxic proteins was found to be effective in cell culture on cancer cells. To combine these two properties, it is necessary to optimally engineer solid tumor-colonizing bacteria to stably encode cytotoxic cargo for days or even weeks in vivo. Due to regulatory requirements, the use of classical antibiotic resistance to maintain a foreign plasmid in bacteria is unacceptable. Thus, in mouse allograft studies, we evaluated a plasmid maintenance system that does not include antibiotic resistance. This system is based on the chromosomal deletion of an essential gene (such as aspartate semialdehyde dehydrogenase, asd) and the encoding of this gene on a heterologous plasmid to support bacterial growth. Complementation of an asd deletion for plasmid maintenance has been demonstrated previously ⁵³ , although difficulties with reproducibility and persistence over several days have been reported ⁵⁴ . The present inventors adapted this system for use in Y. enterocolitica AyopH,O,P,E,M,T, in which the present inventors further deleted the chromosomally encoded asd (resulting in Y. enterocolitica AyopH,O,P,E, M,T Aasd), which was then returned to the medium copy number plasmid, pBad-MycHisA (pBad-MycHisAasd). The asd gene was cloned from Y. enterocolitica 8081 and inserted into the PciI site of pBad-MycHisA in forward and reverse orientation, respectively, with its endogenous promoter and transcription terminator. The growth characteristics of the resulting Y. enterocolitica strains AyopH,O,P,E,M,T Aasd+pBadMycHisA-asd (direct or reverse asd orientation) were compared in in vitro culture flasks (BHI medium) with WT and parental Y. enterocolitica strains AyopH,O,P,E,M,T Aasd (Fig. 12). In both orientations, pBad-MycHisA-asd restored the phenotype observed after asd deletion (Fig. 12). In contrast, in a mouse model of B16F10 melanoma, as well as in a 4T1 model, we found that Y. enterocolitica AyopH,O,P,E,M,T Aasd+pBad-MycHisA-asd did not colonize solid tumors to a sufficient extent (Fig. 13 and 14), and from several isolated colonies the authors of the present invention were unable to recover the plasmid pBad-MycHisA-asd. Moreover, colonies isolated from the B16F10 mouse model of melanoma were confirmed by growth on ampicillin-containing plates, which confirmed the presence of the pBad-MycHisA-asd plasmid encoding ampicillin resistance. On day 4 after i.v. injection, only a small fraction of isolated colonies showed resistance to ampicillin (Fig. 13). This result may reflect loss of pBadMycHisA-asd, but may also be due to loss of the ampicillin resistance gene alone or difficulty in restarting ampicillin resistance gene expression quickly enough to avoid killing by ampicillin. In any case, tumor colonization with Y. enterocolitica AyopH,O,P,E,M,T Aasd+pBad-MycHisA-asd, which was observed in the B16F10 mouse model of melanoma, as well as in the 4T1 model, demonstrated a rapid decrease in the number of bacteria in the solid tumor , in contrast to Y. enterocolitica АуорН,О,Р,Э,М,Т. Thus, deletion of chromosomal asd and addition of pBad-MycHisA leads to a sharp decrease in the survival and ability of bacteria to reproduce in vivo. This observation is consistent with reports of decreased viability due to unbalanced asd levels ⁵⁴ .

Endogenous proteins for transport by the T3SS (called Yersinia extrinsic proteins, Yop) are encoded by Y. enterocolitica on a 70 kb plasmid. This plasmid, called the Yersinia virulence plasmid (pYV), additionally encodes the T3SS apparatus. The present inventors evaluated the stability of the pYV plasmid in the 4T1 allograft mouse model. We have successfully isolated pYV from strains collected at day 9 or 10 post infection of mice (FIG. 15). We then performed assays confirming the presence and functionality of the T3SS of the isolated bacterial strains after eight days of growth in a solid tumor in vivo. Thus, the present inventors have considered pYV to be a vector of choice for encoding heterologous cargo for in vivo delivery. Despite this, the present inventors have found that the percentage of bacterial colonies carrying the pYV plasmid is heterogeneous after growth for 9-10 days in 4T1 solid tumors in mice (Fig. 15). In addition to the intrinsic instability of pYV, the Y. enterocolitica strain AyopH,O,P,E,M,T has lost the selective advantage of increasing Yop virulence in vivo.

To stabilize pYV and thus the heterologous cargo encoded on pYV, we adapted the asd system for use in Y. enterocolitica AyopH,O,P,E,M,T on pYV. We have deleted the chromosomally encoded asd (resulting in Y. enterocolitica AuopH,O,P,E,M,T Aasd), which is then returned to pYV (referred to as pYVasd). The asd gene was cloned from Y. enterocolitica 8081 (Y. enterocolitica subspecies enterocolitica 8081; NCBI reference sequence: NC_008800.1) and inserted by homologous recombination into pYV (in the natural insertion region upstream of SycO) with its endogenous promoter and transcription terminator. The growth characteristics of the resulting Y. enterocolitica strains AyopH,O,P,E,M,T Aasd+pYVasd were compared in in vitro culture flasks (BHI medium) with WT and parental Y. enteroco strains

- 66 044058 litica AyopH,O,P,E,M,T Aasd (Fig. 12). pYV-asd was able to restore the phenotype observed after asd deletion (Fig. 12), although the restoration was not complete and a slight reduction in in vitro growth could be observed. In contrast, in an in vivo syngeneic mouse model of 4T1 cancer, we found that Y. enterocolitica AuopH,O,P,E,M,T Aasd+pYV-asd efficiently colonizes solid tumors (Fig. 16). Strikingly, all colonies isolated from solid tumors at days 9–10 post-injection were found to still contain the pYV plasmid (selection on arsenite-containing growth plates; arsenite resistance is associated with the presence of arsRBC genes on pYV ⁵⁵ ) (Fig. . 15). Therefore, pYV-asd was unexpectedly demonstrated to be a stable in vivo vector for encoding heterologous proteins to be expressed in solid tumors by bacterial colonization within days and weeks in the original Y. enterocolitica strain AuopH,O,P,E,M. T Aasd.

Efficacy of Y. enterocolitica AHOPEMT in delaying tumor progression and the impact of altering VirF activity as well as increasing stability.

Experiments similar to those performed with 4T1 cells (Figs. 9 and 10) were performed in the EMT6 mouse model of breast cancer, in which wild-type Balb/C mice s.c. infused with EMT6 breast cancer cell allograft and treated with a single i.v. introduction of bacteria when the tumor reached a size of approximately 80-250 mm3. Day v.v. bacterial injections were designated as day 0, all mice received i.p. injection of Desferal one day before D0. Treatment of Y. enterocolitica with AHOPEMAT had no effect on tumor progression compared with saline. Y. enterocolitica AHOPEMAT pYV-YopE1. ₁₃₈ -(BH3 tBID)2 showed a minor effect on tumor progression, which was enhanced by Y. enterocolitica AHOPEMAT AHairpinI-VirF pYV-YopE1. ₁₃₈ -(BH3 tBID)2 (Fig. 45). These results highlight that such bacteria and their T3SS can be used to interfere with tumor progression, and that manipulation of VirF activity can be used to modulate bacterial T3SS activity after administration in vivo. Moreover, the use of Y. enterocolitica AHOPEMAT AHairpinI-VirF Aasd pYV-asd-YopE1_ ₁₃₈ -(BH3 tBID)2 further enhances the effect on tumor progression (Fig. 45), highlighting the benefits of enhanced genetic stability after systemic administration.

Delivery of RIG-1-like receptor pathway trigger proteins by bacterial T3SS to induce type I IFN response.

Cytosolic nucleic acids are sensed by a receptor such as members of the RIG1‐like receptor (RLR) family, which detect RNA from the pathogen in the cytosol ⁵⁶ . RIG‐1 and MDA5 consist of two N‐terminal CARD domains and a central (DExD/H) helicase domain that senses specific nucleotides ⁵⁶ . Binding to stimulatory RNA induces structural rearrangements in RIG-I (and MDA5), which release the CARD for subsequent binding to unanchored K63-linked ubiquitin chains to form oligomers ⁵⁶ (and in the case of MDA5, to form filaments ⁵⁶ ). The oligomerized CARD domains of RIG-I and MDA5 interact with the CARD domain of MAVS. This interaction stimulates polymerization of the single CARD domain of MAVS, which induces downstream signaling, ultimately leading to the induction of type I IFN genes ⁵⁶ .

The present inventors have obtained bacterial strains expressing two N-terminal CARD domains of human or mouse-derived RIG-1 fused to an N-terminal bacterial secretion signal for delivery by a T3SS, specifically YopE ₁ . ₁₃₈ (SEQ ID NO: 37 and 38). Delivery of the _{YopE1_138} - CARD2 RIG-1 fusion protein was assessed using a standard in vitro secretion assay, and the functionality of the delivered proteins was assessed in a reporter cell line for type I IFN induction. B16F10 mouse melanoma reporter cells for type I IFN stimulation rely on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with different amounts (MO) of bacterial strains expressed from a plasmid derived from pBadMycHisA (pBad_Si2) and translocating the YopE1_ ₁₃₈ - CARD2 RIG-1 protein. The N-terminal CARD domains of mouse and human RIG-1 demonstrated the induction of a dose-dependent type I IFN response in the reporter cell line (Fig. 17), while the background bacterial strain (Y. enterocolitica AHOPEMAT) was unable to induce such a response (Fig. 17). The human and mouse RIG-1 CARD domains induced a similar type I IFN response in a mouse reporter cell line (Fig. 17), consistent with a high degree of sequence identity (76%) and similarity (88.5%). Thus, fusion with the N-terminal secretion signal of bacteria led to the successful delivery of human and mouse YopE1_ ₁₃₈ - CARD2 RIG-1 proteins expressed in bacteria and did not prevent the folding and function of the RIG-1 CARD domains in the eukaryotic cell. This means that RIG1 CARD domains fused to YopE are still able to multimerize and induce MAVS multimerization, which is unexpected.

In the following experiments using this type I IFN reporter cell line B16F10, we compared Y. enterocolitica AHOPEMAT with Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid (pBad_Si2) YopE1. ₁₃₈ -MycHis or YopE1_ ₁₃₈ - 67 044058

CARD2Rig1 person. Again, delivery of human RIG-1 CARD domains induced a dose-dependent type I IFN response, whereas Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT delivering

YopE1. ₁₃₈ -MycHis had no effect on the type I IFN response (Fig. 18). In the same assay, we compared the type I IFN induction potential of bacteria delivering domains

CARD RIG-1, with positive control, mouse interferon gamma (IFN).

Quite surprisingly, it was found that bacterial delivery of RIG-1 CARD domains was able to induce a maximal response in the reporter cell line, similar to that obtained in the positive control for type I IFN induction, IFNu (FIGS. 18 and 19).

In the following experiments, we infected 4T1 mouse breast cancer cells or B16F10 melanoma cells with WT and transferred the supernatant, presumably containing IFN, 4 hours later to the B16F10 type I IFN reporter cell line. In this manner, the present inventors compared Y. enterocolitica AHOPEMAT with Y. enterocolitica AHOPEMAT encoding YopE1 on an endogenous virulence plasmid (pYV). ₁₃₈ - CARD2RIG1 mouse. Delivery of pYV-encoded mouse RIG-1 CARD domains induced a dose-dependent type I IFN response, whereas Y. enterocolitica AHOPEMAT had no effect on the type I IFN response in B16F1O WT (Fig. 20) or 4T1 (Fig. 21) cells.

The next experiment assessed the potential of several versions consisting of different lengths of mouse RIG-1 CARD to induce a type I IFN response. The CARD domains of RIG-1 are predicted to be encoded by amino acids 1-172 (mouse sequence, Uniprot no. Q6Q899). The present inventors evaluated YopE ₁ . ₁₃₈ -domains CARD1-246 of mouse RIG1, YopE ₁ . ₁₃₈ -domains CARD1.229 of mouse RIG1 and YopE ₁ . ₁₃₈ CARD1 domains. ₂₁₈ mouse RIG1 on B16F10 melanoma IFN reporter cells, as well as RAW macrophage IFN reporter cells (Fig. 27-28). It was found that YopE ₁ . ₁₃₈ domains CARD ₁ _ ₂₄₆ mouse RIG1, YopE ₁ . ₁₃₈ ^CARD domains 1-229 of mouse RIG1 and YopE ₁ . The ₁₃₈ ^CARD domains 1-218 of mouse RIG1 are equally active. In the next experiment, the present inventors evaluated the biological activity of bacterially delivered MDA5. We have cloned several versions consisting of different lengths of mouse MDA5 CARD and assessed their biological activity in inducing type I IFN responses in B16F10 IFN reporter cells. The CARD domains of MDA5 are predicted to be encoded by amino acids 1-190 (mouse sequence, Uniprot no. Q8R5F7). The inventors of the present invention have evaluated

YopE ₁ . ₁₃₈ -domains CARD1.294 of mouse MDA5 and YopE ₁ . ₁₃₈ -domains of mouse CARD1.231 MDA5 on IFN reporter B16F10 melanoma cells. All variants were found to be active (FIG. 29). Surprisingly, it was found that the activity of the delivered MDA5 CARDs was much less high than the RIG-1 CARDs, even though the proteins had very similar biological function and a protein structure consisting of two N-terminal CARD domains and a central (DExD/H ) helicase domain that senses specific nucleotides ⁵⁶ .

Delivery of cGAS/STING pathway trigger proteins by bacterial T3SS to induce type I IFN response.

In the cGAS/STING pathway, cytosolic double-stranded DNA is found through binding to the enzyme cyclic GMP-AMP synthase (cGAS). After binding to dsDNA (double-stranded DNA), cGAS is activated and produces a second messenger cyclic dinucleotide, cyclic GMP-AMP (cGAMP). cGAMP directly binds to the endoplasmic reticulum receptor protein STING (Stimulator of IFN Genes, stimulator of IFN genes). Upon binding to cGAMP, STING is activated and induces a signal transduction pathway leading to the transcription of type I IFN and other coregulated genes ⁵⁷ . Human CGAS produces 2',3'-cGAMP (containing 2'-5' and 3'-5' phosphodiester linkages), but other cyclic dinucleotides have been shown to induce mouse or human STING at varying levels. Such cyclic dinucleotides include 3',3'cGAMP (e.g., produced by Vibrio cholera DncV or some eukaryotic cGAS), cyclic di-AMP (e.g., produced by CdaA or DisA of various Gram-positive species), or cyclic di-GMP (e.g., produced by Pseudomonas aeruginosa WspR ) ^57.58 . While human WT STING (and mouse STING) recognizes 2′,3′‐cGAMP, 3′,3′‐cGAMP, cyclic diAMP and cyclic di‐GMP, some natural variants of human STING respond differently to these agonists ⁵⁹ . To activate the cGAS/STING pathway after bacterial delivery of proteins, we cloned the expressible cyclic di-GMP-producing P. aeruginosa WspR and delivered it to Y. enterocolitica via T3SS. To increase the activity of WspR, only its GGDEF domain (diguanylate cyclase domain) was used, and the stem domain located in the 3′-5′ direction was replaced with the leucine zipper motif GCN4 from yeast. Dimerization is known to be required for WspR activity to occur, and the leucine zipper GCN4 has been demonstrated to form parallel double helices and thus act as a potent demerization module. The GCN4 motif was fused to the GGDEF domain of WspR, including a natural linker between GGDEF and the helical stem, to provide interdomain flexibility comparable to wild-type WspR ⁶⁰ .

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Delivery of the _YopE1-138 -leucine zipper GCN4-GGDEF domain WspR (abbreviated: YopE _1-138 -WspR) fusion protein (SEQ ID NO: 39) was assessed in a reporter cell line for the induction of type I IFN. B16F10 mouse melanoma reporter cells for type I IFN stimulation rely on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with different amounts (MO) of bacterial strains expressed from a plasmid derived from pBadMycHisA (pBad_Si2) and translocating the _YopE1-138 -WspR protein. The GGDEF domain of P. aeruginosa WspR fused to the leucine zipper motif of GCN4 demonstrated dose-dependent induction of a type I IFN response in the reporter cell line (Fig. 22), while the background bacterial strain (Y. enterocolitica AHOPEMAT) was unable to induce such a response. (Fig. 22). Thus, fusion with the N-terminal secretion signal of bacteria led to the successful delivery of the bacterially expressed protein _YopE1-138 -leucine zipper GCN4 (yeast)-GGDEF WspR (P. aeruginosa) and did not prevent the folding and function of this three-part protein in eukaryotic cell. This means that YopE leucine zipper fused GCN4 - GGDEF WspRs are still able to dimerize and thus result in active GGDEF domains, which is unexpected.

For the following experiments, we cloned V. cholerae DncV (producing 3′,3′-cGAMP) ⁵⁷ , Bacillus cereus DisA-like protein (producing cyclic di-AMP) ⁶¹ and eukaryotic anemone (Nematostella vectensis) cGAS (producing 3′, 3'-cGAMP) ⁵⁷ , which have been reported to be active in the absence of external stimuli for expression and translocation by bacteria. DisA-type cyclases typically form octamers ⁶¹ which may be incompatible with N-terminal YopE fusion and bacterial delivery. The B. cereus DisA-like protein (PDB code 2fb5) was identified based on structural similarity to the diadenylate cyclase (DAC) domain of classical DisA proteins 61, but notably lacks all of the helices that other DisA proteins are known to require for multimerization . The present inventors therefore decided to take advantage of the putatively monomeric active DisA-like protein from B. cereus (PDB code 2fb5; residues 76-205).

Delivery of V. cholerae YopE1- ₁₃₈ - DncV fusion protein (SEQ ID NO: 41), B. cereus YopE1- ₁₃₈ - DisA-like protein (SEQ ID NO: 42) and YopE1- ₁₃₈ - anemone cGAS (SEQ ID NO: 43 ) was assessed in a reporter cell line for type I IFN induction. B16F10 mouse melanoma reporter cells for type I IFN stimulation rely on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with different quantities (MZ) of bacterial strains expressed from a pBadMycHisA-derived plasmid (pBad_Si2) and translocating YopE _1-138 DncV V. cholerae, _YopE1-138 - DisA-like protein of B. cereus and YopE1- ₁₃₈ - anemone cGAS. V. cholerae _YopE1-138 -DncV, B. cereus _YopE1-138 -DisA-like protein, and anemone _YopE1-138 -cGAS all demonstrated dose-dependent induction of a type I IFN response in a reporter cell line (Fig. 23), while background strain of bacteria (Y. enterocolitica AHOPEMAT) or Y. enterocolitica AHOPEMAT delivering YopE1-138 _- MycHis were not able to induce such a response (Fig. 23). Anemone (Nematostella vectensis) cGAS, which produces 3',3'-cGAMP, showed the highest activity, while DncV of V. cholerae (producing 3',3'-cGAMP) and DisA-like protein of Bacillus cereus (producing cyclic di-AMP) activate the type I IFN response to a similar extent.

Thus, fusion with the N-terminal secretion signal of bacteria led to the successful delivery of the bacterially expressed proteins YopE1-138-DncV V. cholerae, _YopE1-138 -DisA- _like protein B. cereus and _YopE1-138 -cGAS anemone and did not prevent folding and the function of these proteins in a eukaryotic cell, which could not be predicted.

Alternatively, mouse IRF3, a central transcription factor that functions downstream of RLR‐ or cGAS/STING‐dependent signaling ⁶² , has been cloned for expression and transport in bacteria. In the absence of activation, IRF-3 resides in a latent conformation in the cytoplasm. Only after activation of upstream receptors such as RIG-1, MDA5 or STING, IRF-3 is phosphorylated by TBK1 and IKKε and thus activated. Phosphorylation of IRF‐3 leads to dimerization, nuclear translocation and binding to coactivators ⁶² . To obtain a constitutively active version of IRF3, we replaced one of the most important phosphorylation sites (Ser397 in mouse IRF3) with Asp ⁶² . Delivery of the mouse _YopE1-138 -IRF3 Ser397Asp fusion protein (SEQ ID NO: 40) was assessed in an in vitro secretion assay in which secretion of the protein into the surrounding fluid was artificially induced. After protein precipitation with trichloroacetic acid, Western blot analysis with anti-YopE antibody was used to determine secreted amounts of protein (FIG. 24). Although ANOREMT strains encoding mouse _YopE1-138 -BH3 tBID resulted in a strong band in the secreted fraction (at 15-20 kDa), mouse _YopE1-138 -IRF3 Ser397Asp was also found to be secreted, although to a lesser extent. (at the level of 50-75 Da) (Fig. 24). Analysis of the total fraction of bacterial cells revealed that the expression levels of mouse _YopE1-138 -BH3 tBID and mouse _YopE1-138 -IRF3 Ser397Asp are comparable, although for

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Mouse _YopE1-138 -IRF3 Ser397Asp showed a degradation band pattern (Figure 24)

Delivery of cGAS/STING and RIG-1-like receptor pathway trigger proteins via bacterial T3SS to induce type I IFN responses in immune cells.

Delivery of mouse YopE1- ₁₃₈ - CARD2 RIG-1 fusion protein, YopE _1-138 - V. cholerae DncV, B. cereus YopE _1-138 DisA-like protein and anemone YopE1- ₁₃₈ - cGAS fusion protein was assessed in an immune reporter cell line for IFN induction Type I

Mouse RAW264.7 macrophage reporter cells for type I IFN stimulation were based on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with different amounts (MO) of bacterial strains expressed from a pBadMycHisA-derived plasmid (pBad_Si2) and translocating mouse _YopE1-138 -CARD ₂ RIG-1, YopE1. ₁₃₈ -DncV of V. cholerae, YopE1- ₁₃₈ - DisA-like protein of B. cereus and YopE1- ₁₃₈ - cGAS of anemone. Mouse _YopE1-138 -CARD2RIG-1, V. cholerae _YopE1-138 -DncV, and anemone _YopE1-138 -cGAS all demonstrated dose-dependent induction of a type I IFN response in this immune reporter cell line (Fig. 25), while the background bacterial strain (Y. enterocolitica AHOPEMAT) was not able to induce such a response (Fig. 25). Mouse YopE1- ₁₃₈ - CARD2 RIG-1 showed the highest activity, followed by anemone (Nematostella vectensis) cGAS producing 3′,3-cGAMP and V. cholerae DncV (producing 3′,3′-cGAMP). The Bacillus cereus DisA-like protein (producing cyclic di-AMP) was found to only weakly activate the type I IFN response.

For the following experiments, we cloned human cGAS amino acids 161522 (Uniprot No. Q8N884 and SEQ ID NO: 115; producing 2',3'-cGAMP) ⁵⁷ for expression and translocation in bacteria. B16F10 mouse melanoma reporter cells and RAW mouse macrophages for type I IFN stimulation were based on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with varying numbers (MO) of bacterial strains expressed from a pBadMycHisA-derived plasmid (pBad_Si2) and translocating human YopE1- ₁₃₈ - cGAS ₁₆₁ - ₅₂₂ , and demonstrated a dose-dependent induction of a type I IFN response in the reporter cell line, as well as strain bacteria, expressed from a plasmid derived from pBadMycHisA (pBad_Si2) and translocating YopE1_ ₁₃₈ - cGAS anemone, YopE1_ ₁₃₈ - CGAS ₆₀ .4 ₂₂ anemone, YopE1- ₁₃₈ - CdaA ₁₀₁ - ₂₇₃ Listeria, YopE1- ₁₃₈ - DncV V. cholerae or Y opE1- ₁₃₈ DisA-like protein of B. cereus (Fig. 32-33). The most powerful activation was observed for human YopE1_ ₁₃₈ cGAS ₁₆₁ - ₅₂₂ , followed by anemone YopE1- ₁₃₈ - cGAS, anemone YopE1- ₁₃₈ - CGAS ₆₀ - ₄₂₂ . It is interesting to note that the shortest anemone cGAS _{60_422} variant was marginally more active _. YopEi.i3 ₈ - CdaA ₁₀₁ - ₂₇₃ Listeria, YopE1_ ₁₃₈ - DncV V. cholerae or YopE1_ ₁₃₈ - DisA-like protein of V. cereus also showed dose-dependent activation of IFN, although to a lesser extent than cGAS proteins (Fig. 32-33 ).

Delivery of MAVS by a bacterial T3SS to induce type I IFN response.

Cytosolic nucleic acids are sensed by a receptor such as members of the RIG1‐like receptor (RLR) family, which detect RNA from the pathogen in the cytosol ⁵⁶ . RIG‐1 and MDA5 consist of two N‐terminal CARD domains and a central (DExD/H) helicase domain that senses specific nucleotides ⁵⁶ . Binding to stimulatory RNA induces structural rearrangements in RIG-I (and MDA5) that release the CARD for subsequent binding to unanchored K63-linked ubiquitin chains to form oligomers ⁵⁶ (and in the case of MDA5, to form filaments ⁵⁶ ). The oligomerized CARD domains of RIG-I and MDA5 interact with the CARD domain of MAVS. This interaction stimulates polymerization of the single CARD domain of MAVS, which induces downstream signaling, ultimately leading to the induction of type I IFN genes ⁵⁶ .

The present inventors have obtained bacterial strains (based on Y. enterocolitica AHOPEMAT) expressing human-derived N-terminal CARD domains of MAVS fused to an N-terminal bacterial secretion signal for delivery via T3SS, specifically _YopE1-138 . Delivery of the YopE1_ ₁₃₈ - CARD MAVS fusion protein was assessed using a standard in vitro secretion assay, and the functionality of the delivered proteins was assessed in a reporter cell line for type I IFN induction. B16F10 mouse melanoma reporter cells and RAW mouse macrophages for type I IFN stimulation were based on secreted alkaline phosphatase activity under the control of the I-ISG54 promoter, which consists of the IFN-inducible ISG54 promoter enhanced by a multimeric ISRE. Reporter cells were infected with varying amounts (MZ) of Y. enterocolitica AHOPEMAT, expressed from a pBadMycHisA-derived plasmid (pBad_Si2) and translocating the human _YopE1-138 -CARD MAVS protein. The N-terminal CARD domain of mouse MAVS demonstrated a dose-dependent induction of a type I IFN response in a reporter cell line (Figures 30-31), while the background bacterial strain (Y. enterocolitica AHOPEMAT) was unable to induce such a response (Figures 30-31). 31). Human cGAS and mouse RIG-1 CARD domains induced type I IFN responses in a similar manner in mouse reporter cell lines (FIGS. 30-31), with RIG-1 showing the highest activation potential, followed by MAVS and cGAS. Thus, fusion with the N-terminal secretion signal of bacteria led to the successful delivery of the human YopE1_ ₁₃₈ - CARD MAVS protein expressed in bacteria and did not prevent the folding and function of the CARD MAVS domain in the eukaryotic cell. This means that the MAVS CARD domain fused to YopE is still able to multimerize and induce MAVS multimerization, which is unexpected. Even more surprisingly, it was shown that CARD MAVS alone was unable to induce downstream IFN signaling ⁶⁴ , whereupon we discovered that the YopE fusion domain of CARD MAVS potently activates the type I IFN pathway.

Moreover, the C‐terminal transmembrane domain of MAVS has been demonstrated to be essential for MAVS function following DNA transfection, and the CARD domain of MAVS itself has been demonstrated to be inactive when expressed from transfected DNA constructs ⁶⁶ . Moreover, it was demonstrated that the CARD of MAVS fused to a transmembrane region, which has the ability, when expressed from transfected DNA or as a purified protein, to activate the type I IFN response ^64,66 , was dependent on endogenous MAVS for aggregation and thus activation he started ⁶⁴ . The present inventors, using the MAVS KO cell line, were able to demonstrate (FIG. 52) that bacterially delivered YopE CARD fused MAVS is active without endogenous MAVS present on mitochondria. That CARD MAVS fused to YopE is able to multimerize and activate downstream partners without a transmembrane domain and, furthermore, without endogenous MAVS is unexpected.

Comparative analysis with a small molecule STING agonist to induce a type I IFN response in vitro.

Cyclic dinucleotides are well-known agonists of the STING pathway, leading to downstream induction of type I IFN signaling. STING agonists have been described in the literature ⁵⁹ and have been found to primarily act on immune cells, with the highest activity demonstrated on dendritic cells ⁵⁹ . RLR signaling, in contrast, has been found to be more ubiquitously expressed ⁶³ . The present inventors thus compared Y. enterocolitica AHOPEMAT bacteria delivering enzymes that form cyclic dinucleotides (anemone YopE1_ ₁₃₈ -cGAS and human YopE ₁ - ₁₃₈ -cGAS) or bacteria delivering YopE ₁ . ₁₃₈ -domains CARD1_ ₂₁₈ RIG1 mouse, with low molecular weight agonist STING 2'3'-c-di-AM(PS)2 (Rp,Rp) (similar to ADU-S100 from Aduro Biotech) on immune cells (IFN reporter cells RAW macrophages) and non-immune cells (IFN-reporter melanoma B16F1 cells) regarding interferon-inducing potential. On immune cells, a similar activating potential was observed for the low-molecular-weight STING agonist 2'3'-c-di-AM(PS)2 (Rp,Rp) and all three studied protein-delivering bacterial strains (YopE ₁ - ₁₃₈ -cGAS anemone, YopE ₁ - ₁₃₈ human cGAS or YopE ₁ . ₁₃₈ domains of mouse CARD1. ₂₁₈ RIG1), while the bacteria Y. enterocolitica AHOPEMAT, which do not deliver the protein, showed a very weak activating potential (Fig. 34-37). On non-immune cells (cancer cells, melanoma), bacteria-delivered mouse YopE1_ ₁₃₈ CARD1_ ₂₁₈ RIG1 domains, bacteria-delivered anemone YopE1_ ₁₃₈ -cGAS and human YopE1_ ₁₃₈ -cGAS performed equally well and were virtually superior to the small molecule STING agonist, highlighting the more ubiquitous presence of RLRs compared to STING (Figs. 34-37).

Strict T3SS dependence of bacterially delivered CARD domains RIG1 or CARD MAVS.

To prove strictly T3SS-dependent transport, one of the T3SS proteins that forms the translocation pore in the eukaryotic cell membrane (YopB) was deleted. The potential of such yopB-deleted bacteria (referred to as Y. enterocolitica AHOPEMAT-yopB) expressing YopE ₁ . The ₁₃₈ -domains of mouse CARD1_ ₂₄₆ RIG1 or human YopE1_ ₁₃₈ - CARD1_ ₁₀₀ MAVS were assessed in the IFN-reporter macrophage cell line RAW and compared with the yopB-expressing bacteria Y. enterocolitica AHOPEMAT, which also expresses YopE ₁ . _{138 -} domains ^CARD 1-246 of mouse RIG1 or YopE1_ ₁₃₈ - CARD1_ ₁₀₀ of human MAVS (Fig. 38). Although bacteria with wild-type yopB expressing YopE ₁ . ₁₃₈ -domain CARD1_ ₂₄₆ mouse RIG1 or YopE1.138-CARD1.100 human MAVS demonstrated dose-dependent activation of the type I IFN response, yopB-deleted strains expressing these same proteins were unable to induce such a response above the background level due to the background bacterial strain, which did not express the protein to be delivered (Fig. 38). This confirms that YopE ₁ . The ₁₃₈ -domains of mouse CARD1-246 RIG1 or human YopE1- ₁₃₈ -CARD1-100 MAVS were both transported through the T3SS needle into eukaryotic target cells.

Induction of type I IFN response in a crude tumor isolate/

To confirm that the type I IFN response can be initiated in the tumor microenvironment, we analyzed crude tumor isolates infected ex vivo with bacterial strains, followed by interferon beta ELISA. Wild-type Balb/C mice with s.c. allograft EMT6 breast cancer cells were killed when the tumor reached volume. Tumors were ground, split and seeded as a suspension of individual cells for 24

- 71 044058 well plates. Such cells from two different tumors were left uninfected (dashed lines in Fig. 39) or infected with Y. enterocolitica AHOPEMAT or Y. enterocolitica AHOPEMAT encoded on the pBadMycHisA-derived plasmid YopE ₁ . ₁₃₈ -domains CARD ₁ _ ₂₄₆ mouse RIG1. IFN stimulation was assessed using an interferon beta ELISA and it was demonstrated that although Y. enterocolitica AHOPEMAT was unable to induce interferon beta secretion, infection with Y. enterocolitica AHOPEMAT encoding YopE ₁ . The ₁₃₈ -domain CARD1.246 mouse RIG1 resulted in dose-dependent secretion of interferon beta by a crude tumor isolate of two different tumors (Fig. 39). This confirms that bacterially delivered RIG1 CARD domains are capable of inducing interferon production in mixed cell populations consisting of cancer cells, immune cells, and all other cells in the tumor microenvironment.

Efficacy of Y. enterocolitica AHOPEMAT delivering CARD RIG1 or cGAS in delaying tumor progression/

To assess the impact of YopE ₁ . ₁₃₈ -domains ^CARD 1-246 mouse RIG1 and human YopE ₁ _ ₁₃₈ -cGAS delivered to tumor cells in vivo, the authors of the present invention conducted a study on wild-type Balb/C mice with p.c. allograft of EMT6 breast cancer cells. Mice were injected intratumorally (i.t.) with PBS (Fig. 40) or 7.5x107 Y. enterocolitica AHOPEMAT, Y. enterocolitica AHOPEMAT+YopE _1-138 mouse CARD1.246 RIG1 domains or Y. enterocolitica AHOPEMAT+YopE _1-138 cGAS person when the tumor reached a size of approximately 60-130 mm ³ . Day one v.o. bacterial injections were designated as day 0. Mice were injected i.o. injections on D0, D1, D5, D6, D10 and D11. Tumor volume was measured over the following days using a caliper. Treatment of Y. enterocolitica with AHOPEMAT alone demonstrated an effect on tumor volume progression, with 4/14 mice experiencing complete tumor regression (FIG. 41). Y. enterocolitica AHOPEMAT, which delivers a type I IFN response-inducing protein, either CARD RIG1 or cGAS, was found to result in a greater effect on tumor progression; 8/14 (CARD RIG1) or 8/15 (cGAS) mice experienced complete and long-term tumor regression (Fig. 42-43). These results highlight that such bacteria and their T3SS can be used to very significantly inhibit tumor progression, and that delivery of type I IFN-inducing proteins is well suited for inducing regression of the primary tumor.

Mice with complete tumor regression were then followed until day 65 after initial tumor allografting, after which, on day 65, antigen challenge was repeated with EMT6 breast cancer cells on the contralateral side to assess immune-mediated memory and systemic activity against these cancer cells. In this antigen rechallenge study, no additional treatment was administered, and the mice were simply monitored for tumor progression on the contralateral side and compared with naïve mice (mice that had not previously been exposed to EMT6 breast cancer cells, but all other parameters which, such as age, were identical). It was found that while in mice not previously used in experiments, s.c. allografted tumor cells resulted in tumor growth, all EMT6 tumor-pretreated mice with complete regression on the contralateral side were protected from tumor growth (FIG. 44). It is noteworthy that tumors in mice with previous complete regression induced by bacterial treatment on the contralateral side began to grow within a few days and reached volumes of up to >100 mm ³ (with a maximum volume at approximately day 10 after the second transplantation) and then shriveled (Fig. 44). This lag period may indicate an adaptive immune system response that takes several days to fully establish.

In the following experiments to evaluate the effect of YopE ₁ . ₁₃₈ ^-CA domains RD1-246 mouse RIG1 and human YopE1_ 138-cGAS delivered to tumor cells in vivo, the present inventors conducted a study on wild-type C57BL/6 mice with p.c. allograft of B16F10 melanoma cancer cells. Mice were injected intratumorally (i.t.) with PBS or 7.5x10 ⁷ Y. enterocolitica AHOPEMAT, Y. enterocolitica AHOPEMAT+YopE1.138 mouse CARD1-246 RIG1 domains, or human Y. enterocolitica AHOPEMAT+YopE1.138 cGAS when the tumor reached size approximately 75 mm ³ . Day one v.o. bacterial injections were designated as day 0. Mice were injected i.o. injections at D0, D1, D2, D3, D6 and D9. Tumor volume was measured over the following days using a caliper. Treatment of Y. enterocolitica with AHOPEMAT alone demonstrated an effect on tumor volume progression, with 1/15 mice experiencing complete tumor regression (FIG. 46). It was found that Y. enterocolitica AHOPEMAT, which delivered a type I IFN response-inducing protein, either CARD RIG1 or cGAS, resulted in a very pronounced effect on tumor progression; 5/15 (CARD RIG1) or 8/15 (cGAS) mice experienced complete and long-term tumor regression (Fig. 46). These results highlight that such bacteria and their T3SS can be used to very significantly inhibit tumor progression, and that delivery of type I IFN-inducing proteins is well suited for inducing regression of the primary tumor. It is noteworthy, especially in the case of bacteria delivering human YopE1_ ₁₃₈ -cGAS, that immediately after the first administrations there was an increase in tumor volume compared to PBS-treated controls, which may be caused by the migration of leukocytes into the tumor (pseudoprogression) caused by intracellular delivery cGAS inducing protein

Type I IFN.

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Claims (14)

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MAVS forms functional prion-like aggregates to activate and propagate antiviral innate immune response. Cell. 146:448-461. 65 Kranzusch, P.J., A.S. Lee, J.M. Berger, and J.A. Doudna. 2013. Structure of human cGAS reveals a conserved family of second-messenger enzymes in innate immunity. Cell Rep. 3:1362-1368. 66 Seth, R.B., L. Sun, C.K. Ea, and Z.J. Chen. 2005. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF 3. Cell. 122:669-682. CLAIM 1. A recombinant virulence-attenuated gram-negative bacterial strain containing a nucleotide molecule that contains a nucleotide sequence encoding a heterologous protein fused in frame with the 3'-end of a nucleotide sequence encoding a delivery signal from a bacterial effector protein of the type III secretion system (T3SS), wherein said nucleotide sequence encoding a delivery signal from a bacterial effector protein is operably linked to a promoter, and wherein said heterologous protein is a protein involved in the induction or regulation of an interferon (IFN) response, wherein said heterologous protein is involved in the induction or regulation of an IFN I response type, selected from the group consisting of the RIG-I-like receptor (RLR) family, other CARD domain-containing proteins, - 76 044058 involved in antiviral signaling and induction of type I IFN, and enzymes forming cyclic dinucleotides, such as cyclases cyclic di-AMP, cyclic di-GMP and cyclic di-GAMP, selected from the group consisting of WspR, DncV, DisA and DisA-like protein, CdaA, CdaS and cGAS, which lead to STING stimulation. 2. A recombinant virulence-attenuated gram-negative bacterial strain according to claim 1, characterized in that the specified protein involved in the induction or regulation of the IFN response is a protein involved in the induction or regulation of the type I IFN response, and the specified protein involved in the induction or regulation of the type I IFN response, is selected from the group consisting of RIG1, MDA5, MAVS/IPS-1, WspR, DncV, DisA and DisA-like protein, CdaA and cGAS or fragments thereof. 3. A recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 1 and 2, characterized in that said recombinant virulence-attenuated gram-negative bacterial strain additionally contains a deletion of a chromosomal gene encoding an endogenous protein necessary for growth, and an endogenous virulence plasmid containing a nucleotide sequence that contains a gene encoding the specified endogenous protein necessary for growth, operably linked to a promoter. 4. A recombinant virulence-attenuated gram-negative bacterial strain according to claim 3, characterized in that said recombinant virulence-attenuated gram-negative bacterial strain is deficient in the production of at least one bacterial effector protein. 5. A recombinant gram-negative strain of bacteria attenuated in virulence according to claim 3 or 4, characterized in that said gene encoding an endogenous protein necessary for growth is selected from a gene encoding an enzyme necessary for the production of amino acids, a gene encoding an enzyme involved in biosynthesis of peptidoglycan, a gene encoding an enzyme involved in the biosynthesis of LPS (lipopolysaccharide), a gene encoding an enzyme involved in nucleotide synthesis, and a gene encoding a translation initiation factor. 6. A recombinant virulence-attenuated gram-negative strain of bacteria according to any one of claims 3-5, characterized in that said gene encoding an endogenous enzyme necessary for growth is a gene encoding an enzyme necessary for the production of an amino acid, and said enzyme necessary for for amino acid production, is aspartate beta-semialdehyde dehydrogenase (asd). 7. A recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 1 to 6, characterized in that said recombinant virulence-attenuated gram-negative bacterial strain is a Yersinia strain. 8. A recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 3-7, characterized in that said gene encoding an endogenous enzyme necessary for growth located on an endogenous virulence plasmid contains its endogenous promoter and its endogenous transcription terminator. 9. A recombinant virulence-attenuated gram-negative bacterial strain according to claim 8, characterized in that said gene encoding an endogenous enzyme necessary for growth, its endogenous promoter and its endogenous transcription terminator are located at a distance of 122 bp. in the 3'-5' direction from the start of orf155 (SycO) on the endogenous virulence plasmid. 10. Recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 1-9, characterized in that said recombinant virulence-attenuated gram-negative bacterial strain additionally contains modulation in the thermosensitive region of RNA in the direction 3'-5' from the gene encoding endogenous DNA -binding protein of the AraC type. 11. Recombinant virulence-attenuated gram-negative bacterial strain according to claim 10, characterized in that said modulation in the thermosensitive region of RNA in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type contains a deletion that eliminates the hairpin structure RNA or parts thereof in the 3'-5' direction from the gene encoding the endogenous DNA-binding protein of the AraC type. 12. Recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 10 and 11, characterized in that said DNA-binding protein of the AraC type is VirF. 13. A recombinant virulence-attenuated gram-negative bacterial strain according to any one of claims 1 to 12, characterized in that said recombinant virulence-attenuated gram-negative bacterial strain is Yersinia enterolitica. 14. A method of treating cancer in a subject, comprising administering to said subject a recombinant virulence-attenuated gram-negative strain of bacteria according to any one of claims 1 to 13, wherein said recombinant virulence-attenuated gram-negative strain of bacteria is administered in an amount that is sufficient to treat the subject. -