Kynurenine responsive bacteria and cancer therapies

Abstract

Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence (e.g., not endogenous to said bacteria) encoding a kynurenine transcriptional regulator protein, and ii) a second nucleotide sequence (e.g., not endogenous to said bacteria or overexpressing an endogenous equivalent protein) encoding a kynurenine transporter protein. In particular embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload.

Description

[0001]Attorney Docket Number: CCF-42779.601 KYNURENINE RESPONSIVE BACTERIA AND CANCER THERAPIES The present application claims priority to US Provisional application serial number 63 / 550,147, filed February 6, 2024, and which is herein incorporated by reference in its entirety, specifically including all of the figures. SEQUENCE LISTING PARAGRAPH The text of the computer readable sequence listing filed herewith, titled “CCF_42779_601_SequenceListing.xml”, created February 4, 2025, having a file size of 20,319 bytes, is hereby incorporated by reference in its entirety. This invention was made with government support under CA267711 and CA043703 awarded by the National Institutes of Health. The government has certain rights in the invention. FIELD OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence encoding a kynurenine transcriptional regulator protein, and ii) a second nucleotide sequence encoding a kynurenine transporter protein. In particular embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload. BACKGROUND OF THE INVENTION Bacterial-based cancer therapy could be a promising treatment option for solid tumors especially for those cases in which the traditional approaches are inadequate and ineffective. This therapy is based on the high natural tendency of some bacteria like Salmonella enterica, Escherichia coli, and Clostridium novyi to grow specifically within the tumor compared to normal tissues causing both direct toxic effect on the tumor cells and activation of the Attorney Docket Number: CCF-42779.601 immune system to better eradicate the tumor1-3. This preference for the tumor tissues has been attributed to multiple factors including nutrient availability, more protection from immune surveillance and the hypoxic environment within the solid tumors in case of anaerobic bacteria4. With the latest advances in bacterial synthetic biology, the bacterial- based cancer therapy became closer to reality with several clinical trials were completed or are going using genetically modified bacterial strains as either monotherapy or in combination with other therapies including immune checkpoint inhibitors (ICIs) (e.g. NCT00004988, NCT01675765, NCT01924689, NCT03435952, etc.). A main limitation, however, for the wide-spread application of the bacterial-based cancer therapy is the need to improve the specificity of the bacteria to the cancer site especially if these bacteria were engineered to carry cytotoxic payload4. With regard to S. enterica, one of the most promising and widely tested bacteria as cancer therapeutic, several previous approaches aimed at attenuating it to reduce its toxicity and improve its tumor targeting5-7. Of note, S. enterica VNP20009 strain which has mutations in purI and msbB genes making it auxotrophic to alanine and less stimulatory of tumor necrosis factor-α production, was among the most promising strains when tested in murine models in terms of specificity to the tumor8. Initial attempts, however, testing VNP20009 in clinical trials found it ineffective in causing tumor regression and that we still need to improve the tumor localization of S. enterica9. This in turn highlights the need to find alternative ways to engineer S. enterica so that we can enhance its safety profile (and specificity to cancer) without reducing its efficacy. SUMMARY OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence (e.g., not endogenous to said bacteria) encoding a kynurenine transcriptional regulator protein, and ii) a second nucleotide sequence (e.g., not endogenous to said bacteria or overexpressing an endogenous equivalent protein) encoding a kynurenine transporter protein. In particular embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a Attorney Docket Number: CCF-42779.601 kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload. In some embodiments, provided herein are compositions comprising: a bacterial cell, wherein the bacterial cell comprises one or more nucleic acid sequences, wherein the one or more nucleic acid sequences comprise: i) a first nucleotide sequence encoding a kynurenine transcriptional regulator protein, ii) a second nucleotide sequence encoding a kynurenine transporter protein, and iii) optionally a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein; and iv) optionally a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein; and v) optionally a fifth nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene important or essential for growth of the bacteria or a gene codes for therapeutic payload. In particular embodiments, provided herein are methods comprising: administering the bacteria above or otherwise described herein to a patient with a tumor or a disease in which kynurenine is accumulating in localized lesions, optionally wherein the patient is a human. In certain embodiments, the administration is such that the bacteria localizes to the tumor, or the localized lesions, of the patient. In further embodiments, the tumor or a localized lesion produces kynurenine and the bacteria preferentially localizes, and / or proliferates in, the tumor or lesion at least in part based on the kynurenine expression. In some embodiments, these bacteria are administered orally, systemically or through direct intratumor, or intra-lesion, injection. In additional embodiments, provided herein are kits or systems comprising: a) the bacteria described above or otherwise herein; and b) a delivery device or composition for administering the bacteria to a patient with a tumor, optionally wherein the patient is a human. In further embodiments, the delivery device or composition is selected from: syringes, oral tablets, capsules, enema, and IV solution. In certain embodiments, the third and / or fourth nucleotide and / or fifth nucleotide sequence is present in the one or more nucleic acid sequences. In particular embodiments, the one or more nucleic acid sequences comprises at least one plasmid sequence or viral vector sequence. In other embodiments, the one or more nucleic acid sequences comprises first and second plasmid (or viral vector) sequences, or are part of the chromosome of the bacteria. In other embodiments, the first plasmid sequence (or viral vector) contains the first and second nucleotide sequences, and wherein the second plasmid (or viral vector) sequence contains the third and / or fourth nucleotide sequences. Attorney Docket Number: CCF-42779.601 In other embodiments, the kynurenine transcriptional regulator protein comprises KynR protein from Pseudomonas aeruginosa and other equivalent KynR proteins from Gram- negative and Gram-positive bacteria, such as those mentioned in Figure 2A and figure 8 or others. In further embodiments, the kynurenine transporter protein comprises an Mtr protein, Tna protein, AroP protein, or their homologues in other bacteria as shown in Figure 9 or other aromatic amino acid transporters which may transport kynurenine such as TyrP, and PheP. In further embodiments, the kynurenine responsive promoter comprises a kynurenine- responsive promoter DNA sequence (Pkyn) or a synthetic sequence designed based on, for example, the consensus sequence described in Figure 9D. In further embodiments, the plasmid replication protein comprises repL or another plasmid replication protein. In other embodiments, the reporter protein is selected from mCherry, luciferase, GFP, tdTomato, CFP, RFP, YFP, β-galactosidase and β-galactosidase. In some embodiments, the bacteria is from the following genera which naturally harbor KynR as shown in Figure 9 or can be engineered in a way equivalent to E. coli including: Salmonella, Escherichia, Shigella, Proteus, other Enterobacteriaceae, Pseudomonas, Cupriavidus, other Gamma proteobacteria, and Bacillus. In particular embodiments, the bacteria is E. coli. In further embodiments, the therapeutic is selected from: immunomodulatory agents, bacterial toxins, and drug-activating enzymes (see, e.g., Chein et al., Current opinion in systems biology 5, 8 (2017); and Gurbatri, et al., Sci Transl Med 12 (2020), both of which are herein incorporated by reference. DESCRIPTION OF THE FIGURES Figure 1. Kynurenine is overproduced in OC and TNBC tumors. A) Kynurenine level in patient TNBC vs healthy breast tissue specimens (n= 5, and 3 for Healthy and TNBC tissue specimens, respectively). B) Kynurenine level in patient high grade serous ovarian cancer (HGSOC) vs benign ovarian specimens (n=7 and 15 for benign ovarian and HGSOC specimens, respectively). C) Syngeneic orthotopic 4T1 TNBC tumors were developed in the mammary fat pads of BALB / c mice, and the tissues were harvested when tumors were ~ 200 mm3. Kynurenine levels in the different tissues were measured through LC-MS / MS (n=3). D) C57Bl / 6J Mice were injected subcutaneously with KPCA.A murine ovarian cancer cells, and tissues were harvested when tumor diameters were ~ 1600 mm3(n=3). All samples were homogenized, diluted, and analyzed through LC-MS / MS. Bars and lines show mean ±SE. Significance was tested using Mann-Whitney test for A-B and using Friedman test for C-D. Figure 2. KynR regulator with its cognate promoter (Pkyn) were cloned on a plasmid system and optimized in E. coli then moved to S. enterica. A) The construct pPaKynR1, in Attorney Docket Number: CCF-42779.601 which KynR with its cognate promoter (PkynB) were cloned from P. aeruginosa upstream of mCherry reporter on a plasmid, showed only modest performance in E. coli, B) Further changes through modifying the ribosomal binding site (RBS) sequence for kynR, and mCherry genes resulted in pPaKynR2-mCherry. C) Mtr transporter was overexpressed on the same plasmid to make pMtr-PaKynR2-mCherry system. D) Expression of the cloned Mtr transporter was tuned to further enhance the sensitivity to kynurenine in p113-Mtr-PaKynR2- mCherry. E) Comparing the KynR-Pkyn cassettes from different bacteria strains for their performance in E. coli K12. All promoter sequences were tested on a plasmid that harbors P. aeruginosa KynR. F) Schematic diagram showing the optimized two-plasmid system (pKynR5-mCherry). pKynR5-mCherry employs kynR gene from P. aeruginosa together with the Mtr transporter on plasmid A. The kynurenine-inducible promoters PCnkynB and PPfkynU from P. fluorescens and C. nectar, respectively, are placed in plasmid B upstream of plasmid replication protein, RepL, and the reporter mCherry. G, H) Performance of the optimized dual-plasmid system, pKynR5-mCherry, in E. coli and S. enterica, respectively. Recombinant E. coli (A -G) or S. enterica (H) harboring each of these plasmids were cultured in M9 media supplemented with glucose 0.4% + / - kynurenine (n=3~6) and mCherry fluorescence / OD600 was measured. Plotted are means ± SE. Figure 3. Engineered S. enterica responds to kynurenine in cancer cell spent media. A) schematic diagram for the whole experiment. B) Murine cancer cell lines (100K cells) were cultured in DMEM-Serum media + / - IFNɣ and spent media were analyzed for kynurenine after 72h. C) The spent media of IFNɣ induced / uninduced cancer cells were aliquoted in 96-well plate. S. enterica / pkynR5-mCherry were then added to each well, and bacterial growth and mCherry fluorescence were monitored. D) 4T1-Luc cancer cells were transduced with either IDO1-exressing vector (IDO1) or control empty vector (EV). E, F) Both cell lines were cultured, and spent media were analyzed for kynurenine and used for culturing engineered S. enterica / pkynR5-mCherry. n=2~4 for each. Plotted are means ± SE. Figure 4. S. enterica was engineered to be dependent on kynurenine for growth. A) Asd and MurI enzymes are essential for biosynthesis of d-glutamate, and diaminopimelic acid, respectively, two key components for bacterial peptidoglycan cell wall formation. B) Schematic diagram for the modified pKynR7 dual plasmid system used to generate S. enterica AD95+ strain (S. enterica ΔmurIΔasd / pkynR7-murI-asd). C) Growth kinetics of the mutant AD95+ in M9 media at at increasing kynurenine concentrations. M9 media were supplemented with glucose 0.4%, and casamino acids 0.1%. D) Both 4T1-IDO1 and 4T1-EV were cultured in DMEM-10% serum media and spent media were aliquoted in 96-well plate. Attorney Docket Number: CCF-42779.601 Kynurenine-controlled S. enterica AD95+ strain was added to different wells and bacteria growth was monitored. Plotted are means ± SE. n=3~4. Figure 5. AD95+ has superior specificity specificity to t KPCA.A tumors compared to VNP20009m. A) Experimental scheme. B, C, D). Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. Once tumors reached an average size of roughly 800 mm3, S. enterica mutants were intraperitoneally injected at a dose of 2~4×106colony forming units (CFU). Mice were euthanized 2 days later, and organs harvested for CFU counting. n=7~10. Experiments were repeated on different days. Graphs depict cumulative data, with each symbol representing results from individual mice. E, F, G) The same experiment was repeated; however, organs were analyzed 7 days after i.p. bacterial injection. H) Experiment was repeated via i.v. injection of bacteria. Lines represent mean ± SE. When no colonies were detected at the highest dilution, the number was stated as 5 CFU / g tissue for statistical analyses which is half the limit of detection. Mann Whitney test was used in statistical analysis. p values are presented in panels. Figure 6. AD95+ cancer specificity depends on tumor kynurenine content. A) 4T1 tumors were established in the mammary fat pads of BALB / c mice. When tumors reached an average size of ~ 500 mm3, S. enterica mutants were intraperitoneally injected at a dose of 2~4×106CFU. Mice were euthanized 2 days later, and organs were harvested for CFU counting. B, C) CFU count in tumor versus spleen and liver for each strain showing engineered AD95+ has significantly higher tumor specificity compared to VNP20009m. D-F) 4T1-IDO1 and 4T1-EV tumors were injected on opposite flanks in NSG mice. When tumors were roughly 300~400 mm3, S. enterica AD95+ or VNP20009 were injected i.p. (2x106cfu). Mice were euthanized 2 days later and kynurenine and number of bacteria in each tumor were determined through LC-MS / MS and plating, respectively. n=7~8 per group. Lines represent mean ± SE. When no colonies were detected at the highest dilution, the number was stated as 5 CFU / g tissue for statistical analyses which is half the limit of detection. Statistical analysis was done by Mann Whitney test. p values are presented in panels. Figure 7. AD95+ attenuates tumor growth. A) Experimental design. KPCA.A tumors were injected subcutaneously in C57BL / 6 mice. Once tumors were detected, S. enterica mutants AD95+ and VNP2009m were i.p. injected at a dose of ~ 2×106CFU weekly. Tumor growth and mice survival were monitored over time. All mice were euthanized 28 days after the tumor injection (when the tumors in the PBS group reached endpoint). n=9 per group. Plotted are means ± SE. B) Tumor growth was monitored weekly via palpation. C) Tumors were harvested at endpoint and mass obtained. D) Spleen weights at endpoint. Plotted are means ± SE. Statistical significance was tested using Kruksal Wallis test. Attorney Docket Number: CCF-42779.601 Figure 8: TCGA database analyses showing kynurenine pathway is upregulated in TNBC compared to healthy breast tissue specimens. A) IDO1 mRNA log2-normalized expression level is increased in TNBC compared to healthy controls in TCGA database. B) TDO2 expression levels are also increased in TNBC. lines show mean ±SE. Significance was tested using Mann-Whitney test. Figure 9: Sequences and gene neighborhood of the cloned kynurenine-responsive promoters. A) Kynurenine is sensed in P. aeruginosa as intermediate metabolite in tryptophan to anthranilic acid metabolic pathway. Kynurenine binding the transcriptional regulator KynR activates transcription from the cognate PkynB promoter. B-C) Structure of the kynurenine metabolizing operons in selected Gram-negative and Gram positive bacteria. The dotted red square shows the sequence in (C) cloned upstream of mCherry and in combination with P. aeruginosa KynR in Figure 2E to make plasmids pKynR-Pa-mCherry, pKynR-Cn-mCherry, pKynR-Pf-mCherry, pKynR-Pa-mCherry, pKynR-Bt-mCherry, pKynR-Bc-mCherry, pKynR-Bp-mCherry, pKynR-Rs-mCherry, and pCerKynR1-mCherry, respectively. All these plasmids harbored P. aeruginosa KynR except for pCerKynR1- mCherry. For pCerKynR1-mCherry, the original B. cereus kynR was incorporated in the plasmid rather than P. aeruginosa KynR since KynR in B. cereus belongs to tetR family regulators unlike KynR in Gram-negative strains, which is Lrp / AsnC family type regulator. D) Sequences highlighted in yellow in (C) were analyzed to find the consensus sequence among the highly responsive Pkyn promoters in Gram-negative bacteria. B. pertussis PkynB was excluded due to poor activity as shown in Figure 2E. B. cereus PkynU was also excluded. The sequence in blue in P. aeruginosa PkynB depicts the predicted -35, -10 and transcriptional start site based on the online BPROM promoter finding tool. For P. aeruginosa PkynB, there was originally an extra 5’ sequence (not shown) which was trimmed in later experiments when found unnecessary for the activity. Figure 10: Comparing AroP, TnaB, and Mtr transporters for their ability to enhance the response to kynurenine in pPakynR2-mCherry plasmid backbone. pPakynR2-mCherry plasmid together with the transporter-containing versions (pMtr-PakynR2-mCherry, pAro- PakynR2-mCherry and pTna-PakynR2-mCherry) were transformed into E. coli. E. coli harboring each of these plasmids were cultured in M9 media supplemented with 0.4% glucose + / - kynurenine at 5, 25, and 100 µM concentrations (n=3) and mCherry fluorescence / OD600was measured. Plotted are means ± SE. Figure 11: Testing pKynR5 dual plasmid system in E. coli and S. enterica using nLuc as a reporter protein. E. coli and S. enterica harboring pkynR5-nLuc dual plasmid system were cultured overnight in M9 media supplemented with 0.4% glucose + / - kynurenine added Attorney Docket Number: CCF-42779.601 at the indicated concentration within 96-well plates and nLuc bioluminescence / OD600was assayed after 24 h. Plotted are means ± SE. n=3. Figure 12: Effect of tryptophan on pKynR5 dual plasmid system performance. E. coli harboring pKynR5-mCherry dual plasmid system were cultured in M9 media supplemented with 0.4% glucose + / - kynurenine at 0 or 5 µM concentrations (n=3) in presence of tryptophan at the indicated concentrations within 96-well plates. mCherry fluorescence / OD600was measured after 9 h. Plotted are means ± SE. Figure 13: Attempts to control S. enterica growth in response to kynurenine by controlling murI and asd gene expression. A and B) Initial attempts aimed to make single gene knockouts in S. enterica (murI or asd gene) and supply this gene in pKynR5 dual plasmid system. C) Both genes were knocked out in one mutant, and they were then supplied together in one operon on plasmid B of pkynR5 dual plasmid system. Growth of the mutants was assessed in M9 media at different kynurenine concentrations. M9 media were supplemented with glucose 0.4%, and casamino acids 0.1%. Plotted are means ± SE of three replicates for each condition. Figure 14: S. enterica metabolizes kynurenine into kynurenic acid. S. enterica ATCC 14028s was cultured overnight in M9 media supplemented with glucose 0.4% and kynurenine 100 µM. The cultures were then centrifuged. Spent media were filtered and analysed for Kynurenine and kynurenic acid concentrations using LC-MS / MS. Figure 15: Both plasmids A, and B are stable in the engineered kynurenine-controlled S. enterica AD95+ strain. Plasmid A carries chloramphenicol-resistance gene cassette while Plasmid B carries kanamycin resistance gene cassette. S. enterica AD95+ was cultured overnight in LB broth supplemented with kynurenine at 20 µM without antibiotics, DAP or d-glutamate. Aliquots of this initial cultures were then diluted 1:100 in fresh LB media supplemented with kynurenine only (Without antibiotics, DAP or d-glutamate). Samples were then taken, centrifuged, and resuspended in PBS at OD 1.0 then plated on LB agar plates supplemented with d-glutamate and DAP with / without addition of antibiotics for colony counting. A) The results confirmed the stability of both plasmids as evidenced by similar number of colonies obtained for plain LB agar plates versus antibiotic-supplemented ones. B) On average OD 1.0 was equivalent to ~ 2 x 108colony forming units (CFU) / ml for all conditions. Figure 16: Wild-type S. enterica ATCC 14028s accumulates at high number in the liver and spleen after intraperitoneal injection. Subcutaneous 4T1 tumors were injected in BALB / c mice. Once tumors reached an average size of ~ 500 mm3, Wild-type S. enterica Attorney Docket Number: CCF-42779.601 ATCC 14028s were i.p. injected at a dose of approximately 2×106CFU. Mice were euthanized 2 days later, and organs were harvested for CFU counting on LB agar plates. Bars represent means ± SE (n=3). Initially, four mice were injected with the bacteria. However, one mouse died prior to the pre-determined endpoint. Figure 17: Parent VNP20009 strain accumulates in tumors at ratios 100~1000-fold higher than other organs after intraperitoneal injection. Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. Once tumors reached an average size of ~600 mm3, S. enterica VNP20009 were i.p. injected at a dose of approximately 2×106CFU. Mice were euthanized 2 days later, and organs were harvested for CFU counting on LB agar plates. Bars represent means ± SE. Figure 18. AD95+ has superior specificity to KPCA.A tumors compared to VNP20009m when injected intravenously. Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. Once tumors reached an average size of roughly 400 mm3, S. enterica mutants were intravenously injected at a dose of ~ 2×106CFU through retroorbital injection. Mice were euthanized 7 days later, and organs harvested for CFU counting (n=5). Each symbol represents results from individual mouse. Lines represent mean ± SE. When no colonies were detected at the highest dilution, the number was stated as 5 CFU / g tissue for statistical analyses which is half the limit of detection. Statistical analysis was done by Mann Whitney test. p values are presented in the panels. Figure 19. AD95+ accumulates at higher levels in tumors compared to VNP20009m when injected directly in subcutaneous tumors. A) Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. Once tumors reached an average size of ~ 600 mm3, S. enterica mutants were intratumorally injected at a dose of approximately 2~4×106CFU. Mice were euthanized 2 days later, and organs were harvested for CFU counting and measuring kynurenine concentration through LC-MS / MS. B, C, D) CFU counts in tumor versus other organs for each strain. E) Kynurenine concentration in the tumor versus liver and spleen, respectively. The experiment was repeated on different days, with n=2~3 for each group. Graphs depict cumulative data, with each symbol representing results from an individual mouse. Bars represent means ± SE. Statistical significance was determined by Mann Whitney test. When no colonies were detected on the agar plates at the highest dilution, the number was stated as 5 CFU / g tissue for the statistical analyses, which is half the limit of detection (10 CFU / g tissue). Figure 20: kynurenine concentration in tumors compared to spleen and liver for experiments in Figures 5 and 6. The LC-MS-MS analyses was performed on tissues harvested 2-days after i.p. bacterial injection. Points represent individual mice (n=6~8 for each group at Attorney Docket Number: CCF-42779.601 each experiment). Bars represent mean ± SE. A) Subcutaneous KPCA.A tumors were injected in C57BL / 6 mice. B) 4T1 breast tumors were injected in the 3rdmammary fat pad in BALB / c mice. C) Subcutaneous tumors were injected in NSG mice on opposite flanks using both 4T1-IDO1 and control 4T1-EV cell lines. Statistical significance was determined by Wilcoxon test. Figure 21: AD95+ is better tolerated than VNP20009m and reduces tumor growth. A) Experimental design. KPCA.A tumors were injected subcutaneously in C57BL / 6 mice. Once tumors were detected, S. enterica mutants AD95+ and VNP2009m were i.p. injected at a dose of ~ 1×107CFU weekly. Tumor growth and mice survival were monitored over time. B) During the experiment, 6 out of 10 mice in the VNP20009m group and 1 out of 10 AD95+ treated mice died. All remaining Mice were euthanized 36 days after the tumor injection (when the tumors in the PBS group reached the endpoint). n=9~10 / group. C) Tumor growth was monitored weekly via palpation. D) Tumors were harvested at endpoint and mass obtained. AD95+ treated mice exhibit significantly smaller tumors. Plotted are means ± SE. E) Images of the excised tumors at the end of the study. Tumors from surviving mice were all harvested on the same day (Day 36 after tumor injection). Fig.22. Maps of the plasmids employed in Example 1. Figure 15A shows the plasmids used in Figures 2A-D. Figure 15B shows the plasmids used in Figures 2E-F. Figure 15C shows the plasmids used in Figure 13. Figure 15D shows the plasmids used in the modified pKynR7 system in Figure 4. Figure 23 shows the nucleic acid sequence of S. enterica asd knockout (SEQ ID NO:1). Figure 24 shows the nucleic acid sequence of S. enterica murI knockout (colony 4) (SEQ ID NO:2). Figure 25 shows the nucleic acid sequence of VNP20009 murI knockout (colony K) (SEQ ID NO:3). DESCRIPTION OF THE INVENTION Provided herein are kits, compositions, systems, and methods for treating a subject with a tumor with kynurenine responsive bacteria (e.g., such that the bacteria reduce or eliminate said tumor). In certain embodiments, the kynurenine responsive bacteria comprise one or more nucleic acid sequences comprising: i) a first nucleotide sequence (e.g., not endogenous to said bacteria) encoding a kynurenine transcriptional regulator protein, and ii) a second nucleotide sequence (e.g., not endogenous to said bacteria or overexpressing an endogenous equivalent protein) encoding a kynurenine transporter protein. In particular Attorney Docket Number: CCF-42779.601 embodiments, the kynurenine responsive bacteria further comprise at least one of the following: i) a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein, ii) a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein, and iii) a fifth or more nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene essential for bacterial growth, or a gene codes a for therapeutic payload. EXAMPLES EXAMPLE 1 Harnessing altered metabolism in solid tumors to enhance tropism of anticancer bacteria Bacterial-based cancer therapy could be a potential alternative treatment for various solid tumors especially for those cases in which the traditional therapies are inadequate. The wide-spread application of this therapy, however, is limited due to the need to increase specificity of the therapeutic bacteria to the cancer area to improve treatment safety and efficacy especially if these bacteria are engineered to synthesize toxins or deliver therapeutic payload to the tumor. We imagined this can be achieved through controlling S. enterica gene expression and growth in response to specific metabolites within the tumor microenvironment. Kynurenine is a catabolite of tryptophan, and is overproduced in solid tumors including OC10-12, breast cancer13-16, colorectal cancer17-19, head and neck squamous cell carcinoma (HNSCC)20and others21creating an immunosuppressive environment that allows the tumor to escape immune surveillance22,23. Kynurenine overproduction in tumors is caused by enhanced expression of IDO1 enzyme (Indoleamine 2,3-dioxygenase 1, the first and rate limiting step in the kynurenine pathway) with some studies suggesting a role for its isozyme TDO2 (Tryptophan 2,3-dioxygenase) which catalyzes the same reaction24,25. In most forms of human cancers, high IDO1 expression is positively correlated with poor prognosis16. Because of that, several IDO1 inhibitors have entered into different phases of oncology clinical trials (e.g. NCT01792050, NCT02077881, and NCT02471846). Aiming at harnessing the altered chemical microenvironment within tumors and specifically the upregulated tryptophan to kynurenine metabolism, we engineered bacterial genetic circuits in Escherichia coli and Salmonella enterica to respond to kynurenine at the Attorney Docket Number: CCF-42779.601 physiological low micromolar levels. Building on these genetic circuits, enabled controlling S. enterica growth in response to kynurenine. Finally, the kynurenine-controlled S. enterica showed superior specificity to the tumors in breast and ovarian cancer murine tumor models when compared to S. enterica VNP20009, one of the most characterized and tumor-specific anticancer strains. RESULTS Kynurenine is enriched in malignant tumors compared to other tissues in both human and preclinical murine models for TNBC and OC The ability to target bacteria to tumors requires controlling the growth of these bacteria in response to a tumor-specific signal. We utilize kynurenine as it is a secreted small molecule enriched in solid tumors. To first confirm if kynurenine pathway is elevated in TNBC, we examined the changes in the expression levels of the key kynurenine producing enzymes, IDO1, and TDO2 in TNBC using the publicly available transcriptomics data in The Cancer Genome Atlas (TCGA). The analysis reveals IDO1 expression is significantly higher in TNBC compared to healthy breast tissue samples (Figure 8A). Likewise, TDO2 expression was also elevated (Figure 8B). We then looked at the kynurenine concentrations in malignant tissues compared to their healthy counterparts. We performed LC-MS / MS spectrometry analyses on a total of 8 independent patient breast specimens (3 TNBC and 5 control (healthy)). Kynurenine levels in TNBC tissue specimens were 16-fold higher compared to the levels in healthy breast tissue (TNBC mean tissue content = 7.5 ± 4.1 nmol / g, n=3; Healthy tissue mean concentration = 0.5 ± 0.1 nmol / g, n=5; p=0.036) (Figure 1A). Likewise, we analyzed a total of 22 patient ovarian tumor specimens (7 benign, and 15 high grade serous OC) from human subjects. We found mean kynurenine content in malignant tumor specimens is significantly higher compared to benign tumor specimens (3.4 ± 1.2 nmol / g vs 0.7 ± 0.1 nmol / g for malignant and benign tumors, respectively, p= 0.01, Figure 1B). For the bacteria to home to the tumor kynurenine, the concentration in tumors should be higher compared to other organs. To test for this hypothesis, we injected the syngeneic TNBC cell line, 4T126,27orthotopically in the mammary fat pad, and the high grade serous murine OC cell line, KPCA.A28subcutaneously (SC) in BALB / c and C57BL / 6 mice, respectively. At endpoint, we harvested multiple tissue and analyzed kynurenine content. Among all tissues examined, kynurenine was highest in tumors with averages of 3.4 ± 1.2 and 3.8 ± 0.4 nmol / g compared to averages of 1.4 ± 0.1 nmol / g and 0.7 ± 0.1 nmol / g in all other tissues, respectively (Figure 1C, D). Collectively, these results indicated that to use Attorney Docket Number: CCF-42779.601 kynurenine as a tumor signal to target therapeutic bacteria, we need to engineer a kynurenine- sensing system in bacteria capable of responding to kynurenine at the low micromolar range (1~10 µM) with high ON / OFF ratio. Leveraging the performance of a natural kynurenine sensing system through plasmid copy number-based signal amplification In the prokaryotic world, some bacteria strains such as Pseudomonas aeruginosa, Burkholderia cepacia and others possess aerobic kynurenine catabolic pathway for tryptophan similar to the one in eukaryotes (40-43). Bacterial kynurenine is produced as an intermediate metabolite before it is further catabolized into anthranillic acid or other metabolites. This kynurenine pathway is regulated through a transcriptional regulator, called KynR40 (44). KynR binds to kynurenine leading to transcriptional activation of downstream enzymes in the pathway (Figure 9). In our experiments, we cloned kynR gene together with its cognate promoter (PkynB) from P. aeruginosa in front of mCherry gene on a plasmid (pPakynR1-mCherry). Transforming this plasmid into E. coli K12 resulted in modest response to kynurenine (2.5-, and 3-fold induction when kynurenine was added to the culture medium at concentration of 1 and 2 mM, respectively) (Figure 2A). Optimizing kynR expression and the ribosomal binding site (RBS) sequence of mCherry resulted in pPaKynR2-mCherry version which showed better response to kynurenine (3.3-fold induction at 50 µM kynurenine concentration) (Figure 2B). We then aimed at further improving the response through overexpressing a transporter to allow for enhanced import of kynurenine. E. coli has three different aromatic amino acid transporters which could serve this function (namely, Mtr, AroP, and TnaB) (45). Expressing each of these three transporters in pPaKynR2-mCherry plasmid found they all enhance the response to kynurenine albeit at different degrees with highest response found for Mtr overexpression (Figure 2C, and 10). Subsequently, we reduced Mtr transporter expression by replacing the upstream medium-strength PJ23115 promoter with the weaker PJ23113 promoter. This resulted in p113Mtr-paKynR2-mCherry version, which demonstrated an induction ratio of 3.1-fold at kynurenine concentration of 5 µM (Figure 2D). In parallel, we also tested kynurenine-responsive promoter sequences from different bacterial strains. P. fluorescens PkynUand Cupriavidus necator PkynBsequences demonstrated improved performance (higher ON / OFF ratio, Figure 2E, 9B-D) compared to PkynB sequence cloned from P. aeruginosa. To maximize the ON / OFF switching ratio of the gene circuit, we adapted a plasmid copy number-based signal amplification technique. P. aeruginosa KynR together with P. Attorney Docket Number: CCF-42779.601 fluorescens PkynU, and C. nectar PkynBcassettes in a dual-plasmid system (pKynR5- mCherry).. The transcriptional regulator, P. aeruginosa KynR, together with Mtr kynurenine transporter (Tra) were placed on one plasmid (Plasmid A) (Figure 2F) while the reporter of interest (mCherry) was placed on a different plasmid (Plasmid B). Plasmid B has two origins of replication (46, 47); the mini-F origin for stable plasmid maintenance, and the P1 phage- derived origin oriL, which is located within the coding region of the replication protein RepL (48). P. fluorescens PkynU was placed upstream of the reporter gene of interest (mCherry) while C. necator PkynBwas placed upstream of plasmid B replication gene (repL) (Figure 2F). When kynurenine is present in the medium, it is actively taken into the bacterial cell through the Mtr kynurenine transporter expressed from plasmid A. Once inside the bacterial cell, kynurenine binds to KynR expressed from plasmid A, then kynurenine-KynR complex activates the transcription of both the reporter protein, and the replication protein (RepL) of plasmid B leading to increase in plasmid B copy number and further increase in the expression of the target reporter protein. Using this system, we reached an ON / OFF ratio of 83- and 87-fold at kynurenine concentrations of 5 µM when initially tested in E. coli and when moved to S. enterica, respectively (Figure 2G-H). Importantly, this system did not respond to kynurenine at 0~1 µM concentration (the typical concentration in plasma)13,29, but shows sharp (exponential) response in the 1~10 µM range (the typical concentration in tumors based on literature 20, 49 and as shown in Figure 1). Similar results were also found in both E. coli and S. enterica when mCherry was replaced by nLuc reporter (Figure 11). Since Mtr serves as tryptophan transporter, we tested how pKynR5-mCherry system behaves in presence of tryptophan. As expected, the results indicated pKynR5-mCherry response to kynurenine is reduced proportionally as the tryptophan concentration in the media is increased (Figure 12). However, even at tryptophan concentration of 50 µM, pKynR5- mCherry system still showed high ON / OFF ratio (16.3-Fold) at 5 µM kynurenine concentration. Engineered S. enterica responds to kynurenine secreted by murine OC and TNBC cell lines. To test the kynurenine level produced by cancer cells in in-vitro cell cultures, OC and TNBC cells were cultured for 72 h with or without IFNɣ (the main inducer for IDO1 enzyme and kynurenine production within tumors). Kynurenine concentration in the culture supernatant was analyzed using LC-MS / MS (Figure 3A). IFNɣ addition induced kynurenine production in the supernatant for each of the tested cell lines, albeit to different extents depending on the cell line (Figure 3B). This included the widely used epithelial OC ID8 cell Attorney Docket Number: CCF-42779.601 line 50, the recently developed KPCA.A and BPPNM which both are genetically defined and establish intraperitoneal tumors recapitulating high grade serous OC 39, and the murine TNBC cell line, 4T1 (ATCC-CRL-2539) (37, 38). We then cultured the engineered S. enterica harboring pkynR5-mCherry using the spent media from each cancer cell line. The results showed that engineered S. enterica responded to kynurenine in the spent media as expected, reflecting the kynurenine concentrations in each sample (Figure 3C). To further validate our kynurenine-responsive genetic circuit, we transduced 4T1 cancer cells with either an ido1-expressing lentiviral vector or an empty vector, creating the 4T1-IDO1 and 4T1-EV cell lines, respectively. Analyzing the culture spent media of both cell lines confirmed the constitutive (without IFNγ induction) production of kynurenine by 4T1-IDO1 cells (Figure 3D, E). When the spent media from both cell lines were used to culture S. enterica / pkynR5-mCherry, mCherry was produced in the 4T1-IDO1 media but not in the 4T1-EV media (Figure 3F). Kynurenine-responsive genetic circuits enabled tuning S. enterica growth in response to kynurenine at the physiological levels present in tumors With pKynR5 dual plasmid system in hand, we used it to control S. enterica growth in response to kynurenine through knocking-out a gene needed for S. enterica growth and supply a copy of this gene under pKynR5 control . We first chose the asd gene which is critical for synthesizing diaminopimelic acid (DAP) needed for making the bacterial peptidoglycan layer (51). Since DAP is not made by mammalian cells, S. enterica will not be able to survive in-vivo without expressing asd gene. Our initial attempts, however, were not very successful as S. enterica was able to grow in minimal media that lacked DAP even without kynurenine supplementation indicating that there is a leak in asd gene expression in the OFF status. Consequently, we further modified our system through modifying the ribosomal binding site (RBS) of the asd gene and including a degradation tag (LAA) at its 3’ end (52). These modifications led to S. enterica Δasd / pkynR5-asd mutant which showed controlled growth in response to kynurenine (Figure 13A). In parallel, we took a similar approach with the murI, gene which is critical for synthesis of d-glutamate (53)30, another monomer needed for making the bacterial peptidoglycan layer and cannot be supplied by the mammalian cells (Figure 13B). To enhance S. enterica dependence on kynurenine, we made a double S. enterica knockout in both murI and asd genes and supplied both genes in an operon on plasmid B . We named this S. enterica mutant, strain AD11+ (Figure 13C). Finally, to further tune the system and reduce the chances of emergence of escaping kynurenine-independent mutants, we optimized the system to supply asd gene on plasmid B, Attorney Docket Number: CCF-42779.601 while supplying murI on plasmid A of the dual plasmid system so that both genes are controlled by kynurenine responsive promoters (Figure 4A, B). We called this improved kynurenine-dependent S. enterica ΔmurIΔasd / pkynR7-murI-asd, Strain “AD95+”. Testing this improved strain in minimal media showed tighter control of the growth in response to kynurenine (Figure 4C) with no growth at 0 µM kynurenine concentration and maximum growth at 2.5~10 µM, the typical concentration we observed in tumors (Figure 1). The reduction in the OD600 observed after the exponential growth phase is presumably due to the lysis of the bacterial cells as a result of consumption of a key nutrient. Previous studies found bacteria like E. coli and Mycobacterium tuberculosis metabolize kynurenine through transaminases into kynurenic acid31,32. Indeed, when wild-type S. enterica was cultured in M9 media supplemented with kynurenine, kynurenine was metabolized into kynurenic acid (Figure 14). Kynurenine consumption may result in inability to synthesize the cell wall monomers (i.e. DAP and d-glutamate) during the active growth phase leading to cell lysis. Finally, the kynurenine-dependent S. enterica AD95+ could grow only in the 4T1- IDO1 spent media (which contained kynurenine) but not in 4T1-EV spent media or fresh DMEM-10% serum media (Figure 4D). Since each of the two plasmids in our system (which harbor kanamycin versus chloramphenicol resistance markers) contains one gene that is needed for survival (asd, and murI), the engineered S. enterica cannot lose either of them. This was confirmed through culturing AD95+ in presence of kynurenine without antibiotics followed by plating on plain versus antibiotic-supplemented plates. The results found similar number of colonies indicating the stability of both plasmids in AD95+ (Figure 15). S. enterica AD95+ shows superior tumor specificity in pre-clinical murine models for OC and TNBC when compared to VNP20009 strain Several previous studies found administering unattenuated wild-type S. enterica leads to severe toxicity and kills the mice within a few days33-35. Consistent with these studies, when wild-type S. enterica ATCC 14028s (Wt) were intraperitoneally (i.p.) injected into 4 subcutaneous tumor-bearing mice, 1 mouse died within 2 days prior to the pre- determined endpoint, while the three remaining mice exhibited signs of severe systemic infection including hunching, grimace, and lethargy. Euthanizing these mice 2 days after bacterial injection, we observed a high number of bacteria in the liver and spleen as expected with tumor to liver ratio of only 159-fold (Figure 16). Consequently, we chose to use S. enterica VNP20009 as a control. VNP20009 strain is a derivative of S. enterica ATCC 14028s which was developed through chemical and UV mutagenesis leading to its Attorney Docket Number: CCF-42779.601 attenuation by purine auxotrophic mutation followed by knocking out of msbB gene in a subsequent study to render its Lipid A less immunogenic36. VNP20009 is one of the best characterized and successful S. enterica mutants in terms of specificity to tumors. Previous studies in mice found VNP20009 accumulates in tumors at numbers up to 1000-fold higher than the numbers in liver, spleen, and other organs8,37. Our experiments testing VNP20009 via intraperitoneal (i.p.) injection in KPCA.A subcutaneous murine tumor models confirmed its preferential accumulation in tumors with tumor / liver ratio close to 1000-fold (Figure 17). To enable more accurate quantification of VNP20009 strain in the tissues, we transformed the bacteria with a chloramphenicol resistance plasmid to generate VNP20009m strain so that it can be plated and counted on agar plates supplemented with chloramphenicol. This was done to reduce the possible false counting contaminating bacterial colonies. To ensure the stability of this chloramphenicol resistance plasmid in VNP20009m, it was supplied with a murI gene copy which was knocked out from VNP20009m chromosome. AD95+ strain likewise contains chloramphenicol resistance plasmid. We compared our engineered AD95+ strain with VNP20009m in subcutaneous KPCA.A tumor models. When the tumors grew to a size of around 600 mm3on average, AD95+ or VNP20009m were injected intraperitoneally at a dose of 2~4×106CFU. Mice were euthanized 2 days later, and tissues were harvested to determine the bacterial distribution in tumors versus liver, spleen, and other organs through plating on LB plates (supplemented with d-glutamate, and DAP) (Figure 5A). The results indicate that both strains proliferate and accumulate in the tumor to similar degrees (Figure 5B). Importantly, AD95+ exhibited higher tumor specificity compared to VNP20009m in terms of lower distribution to other organs with averages of roughly 19,000:1, and 23,000:1, for tumor / liver, and tumor / spleen ratios, respectively (Figure 5C, D). Similar results were also obtained when organs were analyzed at 7 days post- intraperitoneal bacterial injection (Figure 5E-G). To further confirm the superior tumor targeting of AD95+, we opted next to test the distribution after intravenous (i.v.) injection. Compared to i.p. route, i.v. injection leads to faster distribution to distant tissues. The results found i.v. injection of AD95+ (via retro- orbital route) leads again to higher tumor accumulation compared to VNP20009m consistent with i.p. injection at the 2-day timepoint (Figure 5H). The higher tumor specificity for AD95+ was even more evident at 7 days after i.v. injection (Figure 18A-C) with nearly complete absence of AD95+ in all organs except tumors at this time point. This was accompanied by smaller spleen weights when compared to mice injected with VNP20009m (Figure 18D). Finally, direct intratumor injection of both strains confirmed that AD95+ Attorney Docket Number: CCF-42779.601 accumulates in KPCA.A tumors with limited diffusion to other organs in comparison to VNP20009m strain (Figure 19). The two strains were then compared through intraperitoneal injection in orthotopic 4T1 TNBC model injected in the mammary fat pads of BALB / c mice. AD95+ demonstrated again high tumor specificity (Figure 6A-C). Indeed, AD95+ shows high selectivity for tumors even with low kynurenine concentrations (i.e. less than 1 nmol / g, Figure 20). To confirm that AD95+ tumor preference depends on kynurenine, immunocompromised NSG mice were injected with 4T1-IDO1 and 4T1-EV cells on opposite flanks within the same mice. NSG mice have partial impairment in innate immunity and complete ablation of adaptive immunity38. Using NSG mice in this experiment, therefore, minimizes immune-induced kynurenine accumulation in the control 4T1-EV tumors. Both cell lines developed tumors at comparable rates. AD95+ and VNP20009m were injected intraperitoneally to determine relative homing to both tumors. The results indicate AD95+ homing to tumors is dependent on kynurenine with an average of 18-fold difference in accumulation in 4T1-IDO1 versus 4T1-EV tumors (Figure 6D, F). As expected, VNP20009 showed no significant difference in distribution between 4T1-IDO1 and 4T1-EV tumors (Figure 6D, F). Our engineered AD95+ strain retained relatively high specificity to the 4T1- EV tumors (approximately 1000-fold) when compared to its distribution in other organs (Liver, spleen, kidneys, and lungs) (Figure 6D) indicating that the specificity to the tumor is not purely dependent on kynurenine but in part due to also the natural tendency of S. enterica to accumulate in tumors. This also underscores the potential of this engineered strain to target tumors even if their kynurenine concentration is as low as 0.5 nmol / g. Kynurenine-dependent S. enterica AD95+ attenuates tumor growth To investigate if S. enterica AD95+ strain can reduce tumor growth, we established subcutaneous KPCA.A tumors in C57BL / 6 mice. Once tumors were palpable, we administered weekly intraperitoneal injections of either vehicle (PBS) or bacteria at a dose of 1x10⁷ CFU (Figure 21A). Tumor growth was monitored by palpation over a 5-week period.9 out of 10 AD95+ treated mice survived and tolerated the treatment till the endpoint (Figure 21B) equivalent to PBS, albeit with smaller tumors. VNP20009m was evaluated as a benchmark. However, we determined that weekly treatment with VNP20009m at this dose (1x10⁷ CFU) was toxic with only 4 mice surviving to endpoint (Figure 21B). We determined that AD95+ suppresses tumor growth (Figure 21C) and tumor mass compared to PBS treated mice (Figure 21D, E). These studies are consistent with the concept that AD95+ can Attorney Docket Number: CCF-42779.601 specifically home to the tumor and lead to tumor growth inhibition. To further confirm the therapeutic efficacy of AD95+, we repeated the experiment with lower dose (2 x106CFU) for each of AD95+ and VNP20009m (Figure 7A). The results found mice injected with either of the two strains had significantly slower tumor growth and smaller tumors at the endpoint compared to control PBS-injected mice (Figure 7B, C). Nevertheless, mice injected with VNP20009m showed significantly higher degree of splenomegaly at endpoint compared to the PBS or AD95+ injected mice (Figure 7D). These results are consistent with the concept that treatment with AD95+ exhibits lower toxicity to the spleens compared to VNP20009m. Relying on the natural tendency of some bacteria like S. enterica to preferentially accumulate in solid tumors is the core principle of bacterial-based cancer therapy. Despite this natural tropism for tumors, there remains a need to improve tumor bacterial specificity. Previous efforts to engineer tumor-specific bacteria resulted in S. enterica VNP20009, which showed high specificity in murine models (~ 1000-fold tumor : liver ratio)8. VNP20009 was used as a benchmark in our studies. Despite the promising level of tumor site accumulation, this strain was not successful in phase I clinical trials9. Other groups aimed to target tumor hypoxia through using obligate anaerobic bacteria such as Clostridia, which germinate only in anaerobic microenvironments39. These approaches, however, limit the bacterial growth to the tumor core while leaving the outer rim of the tumor unaffected39,40. Here, we sought an alternate unique approach to enhance the specificity of S. enterica to tumors by targeting tumor-enriched kynurenine. A previous study21showed most solid tumors exhibit variable levels of IDO1 overexpression. The proportion of tumor cells with IDO1 overexpression, however, is highly variable between different tumors. In that study, 8 out of 10 ovarian tumor specimens were positive for IDO1 overexpression. In our current study, we found kynurenine production is variable between samples of the same tumor type. In TNBC, 3 / 3 specimens exhibited kynurenine levels more than 4-fold higher than the healthy tissue levels. In OC, 11 / 15 specimens showed high kynurenine levels with 8 / 15 specimens showing kynurenine levels more than 5-fold higher than the average benign ovarian tumor level (Figure 1). With this background insight, we constructed bacterial kynurenine-responsive genetic circuits that allows control of gene expression and growth in the bacteria (including E. coli and S. enterica) in response to kynurenine secreted by solid tumors. Using this circuit, we show high specificity of bacteria to the tumor largely driven by kynurenine. The high specificity of the bacteria to the tumors appears to lead to better tolerance by the mice and less mortality when compared to the benchmark strain, VNP20009 as shown in Figure 21B. Attorney Docket Number: CCF-42779.601 Although we focused here on two cancer types (OC and TNBC), our approach is applicable to other solid tumors as kynurenine accumulation is reported in almost all types of solid tumors17-23. One issue is that kynurenine is not produced equally by all solid tumors and depends on the degree of T-cell and IFNɣ infiltration within the tumor microenvironment 41. Nevertheless, AD95+ mutant could grow to high degrees in the tumors which showed only low kynurenine concentration (0.5 nmol / g) (Figures 5, 6, 19 and 20) with high specificity. Given the heterogeneous nature of solid tumors and the uneven spatial distribution, some studies suggest the actual kynurenine concentration in some locations within the tumor microenvironment is higher than the average kynurenine concentration in the bulk tumor tissue42. This could explain how AD95+ accumulates in tumors with low bulk kynurenine concentration. Our engineered kynurenine-controlled AD95+ strain, and other embodiments herein, may be applied to next generation drug delivery chasse for a variety of therapeutic anti- cancer payloads including enzymes to locally activate chemotherapeutic prodrugs into active drugs43-45, immunomodulatory agents46, and anticancer nanobodies47,48, and as superior alternative to the current tumor-targeting S. enterica strains. Furthermore, applications of engineered kynurenine-controlled genetic circuits as both therapeutics and diagnostics may be extend beyond cancer to other inflammatory diseases in which kynurenine is also overproduced including inflammatory bowel diseases (IBD)49. Additionally, more precise tumor targeting, in certain embodiment, can be achieved through combining kynurenine- based control with other targeting strategies such as purine auxotrophy or hypoxia-and acidity driven bacteria targeting50-52. MATERIALS AND METHODS Table 1: Bacterial plasmids used Plasmid Features Origin / Antibiotic Figure i t 0 0 Attorney Docket Number: CCF-42779.601 PJ23115-AroP P113-Mtr-KynR2-mCherry KynRP.aer.; pBR322 / Cm 2D P h , , Attorney Docket Number: CCF-42779.601 system) oriL / Kan p101-Amp _ Temperature _ iti 11 Bacterial strain Features Figure Escherichia coli K12 (Steller, Primary strain for cloning and testing genetic 2A-G, 10-12 6 Cell line Features Figure 4T1-l Mrin tril n tiv br t n r 13 616 20 Attorney Docket Number: CCF-42779.601 Human breast and ovarian tumor specimen collection. Human ovarian tumor and benign specimens were collected from consented patients undergoing surgery at the Cleveland Clinic Foundation using IRB #19–185. Breast healthy and tumor discarded tissue were obtained from surgical pathology immediately after surgery. The tissues used for this study are not considered essential for diagnosis or treatment decisions and would otherwise be discarded. Tumors were snap frozen and stored for processing. Microbial strains and culturing conditions E. coli Steller (Clontech), S. enterica ATCC 14028s and S. enterica VNP20009 (ATCC BAA-3199, YS1646) were used for all experiments. All were routinely streaked on LB (Lauria Bertani) agar plates and cultured in LB broth. For S. enterica mutants lacking asd and murI genes, daminopimelic acid, and d-glutamate were added to the cultures at 250 µg / ml for each. When needed, ampicillin, kanamycin, and / or chloramphenicol were added at 100, 50, and 17 µg / ml respectively. Cloning kynR and its cognate promoters and optimizing the performance in E. coli. For the initial experiments, the DNA sequence spanning the kynR gene together with its nearby cognate promoter upstream of kynB gene was PCR amplified from P. aeruginosa MRSN1583 and placed upstream of mCherry reporter on plasmid pPaKynR1- mCherry. The RBS sequences were optimized for the kynR and mCherry in pPaKynR2- mCherry using the RBS calculator tool (73,74). To clone the aromatic amino acid transporters Mtr, AroP, and TnaB, each was PCR amplified from E. coli MG1655 genome and placed downstream of PJ23115 promoter to make pMtr-PakynR2-mCherry, pAro- PakynR2-mCherry and pTna-PakynR2-mCherry plasmids. The PJ23115 promoter was then changed for PJ23113 promoter in p113-Mtr-PakynR2-mCherry plasmid. To compare the kynurenine-responsive promoters from various bacteria, the promoter sequence upstream of kynU or kynB gene in each strain was PCR amplified from the corresponding genome and placed upstream of mCherry in a series of plasmids (pKynR-Pa-mCherry, pKynR-Cn- mCherry, pKynR-Pf-mCherry, pKynR-Pa-mCherry, pKynR-Bt-mCherry, pKynR-Bc- mCherry, pKynR-Bp-mCherry, pKynR-Rs-mCherry, pCerKynR1-mCherry). These plasmids contained kynR gene from P. aeruginosa downstream of PJ23114 promoter. In these plasmids, gfp gene was included upstream of each of the tested promoters in an opposite direction. This was done to test for induction of expression in the opposite direction upon Attorney Docket Number: CCF-42779.601 inducing each of the tested promoter sequences with kynurenine. None of the tested promoters induced GFP expression indicating that all tested sequences are unidirectional promoters. Figure 9 shows the sequence of each of the cloned sequences and the consensus kynurenine-responsive promoter sequence. To construct the optimized dual pkynR5-mCherry system, kynR and mtr transporter genes were kept on plasmid A under the control of PJ23113, and PJ23114 promoters respectively (p113m-114kynR) while the reporter mCherry (or nluc gene) was placed in another plasmid (plasmid B, pTrig-cn-repL-pf-cherry). Plasmid B backbone was modified from our previous study (46) through placing C. nectar PkynB promotor sequence upstream of repL gene while P. fluorescens PkynU promoter sequence was placed upstream of mCherry gene. All constructed plasmids were transformed into E. coli Steller through chemical transformation and verified through Sanger sequencing. Transformation of S. enterica was done through electroporation using the purified plasmid. Figure 22 shows the maps of the key constructed plasmids. Tables 1 and 2 show all the plasmids and bacterial strains constructed during the course of the study, respectively. Assessing growth, mCherry fluorescence and nLuc bioluminescence of E. coli and S. enterica. E. coli and S. enterica strains harboring the respective plasmids were cultured in LB broth for 24 h.2 µl of these suspensions were added to 200 µl of M9 media supplemented glucose at 0.4% inside 96-well plates. LB and M9 media were supplemented with chloramphenicol and / or kanamycin when appropriate. The plates were incubated at 37 °C with shaking in Tecan infinite microplate reader. OD600 and mCherry fluorescence (580 nm / 610 nm) were recorded at 30 minute intervals over 24~48 h periods. mCherry fluorescence for each strain was calculated as (mCherry fluorescence of Test strain / OD600) – (Background fluorescence of Control strain / OD600). nLuc bioluminescence was measured after 24 h of incubation using Nano-Glo Luciferase Assay kit (Promega) according to the manufacturer instructions. For experiments assessing the kynurenine-dependent growth of mutant S. enetrica strains, M9 media was supplemented with both glucose at 0.4% and casamino acids at 1%. For these experiments, it was necessary to incubate the LB starter cultures of S. enterica AD95+ for 24 h followed by centrifugation and washing the pellets twice in M9 media. We noticed that traces of LB media allowed growth of S. enterica AD95+ mutant in absence of externally added kynurenine. The suspensions were then diluted 10-fold in M9 media then 2 Attorney Docket Number: CCF-42779.601 µl of these suspensions were added to 200 µl of M9 supplemented with glucose at 0.4% and casamino acids at 1% inside 96-well plates. Knocking out asd and murI genes in S. enterica. For gene knockout in S. enterica ATCC 14028s, bacteria were first rendered ampicillin resistant through transformation with plasmid p101-Amp containing ampicillin- resistance cassette and a temperature-sensitive derivative of pSC101 replication origin.. This plasmid was constructed through deleting λ red recombination genes from pKD46 plasmid53. Two PCR reactions were then used to amplify ~ 1 kb DNA fragments surrounding the sequence to be deleted. The two homologous recombination arms were fused through a third PCR reaction then ligated to a suicide plasmid using the In-Fusion® HD Cloning kit (Clontech). The suicide plasmid contained R6K replication origin, RP4-oriT, sacB as counter selection marker, and kanamycin resistance cassette. The ligated plasmid was transformed into Escherichia coli S17 λpir then transferred to S. enterica through conjugation. The resulted S. enterica merodiploid conjugants conjugants were plated on LB agar plates containing kanamycin at 50 ng / µl and carbenicillin at 200 ng / µL. One S. enterica merodiploid mutant was then selected and re-streaked on LSW-Sucrose agar plate (76) (tryptone 10 g / L, yeast extract 5 g / L, glycerol 5 ml / L, NaCl 0.4 g / l, sucrose 100 g / L and agar 20 g / L) supplemented with kanamycin at 50 ng / µl and either d-glutamate at 250 µg / ml (for murI knockouts) or DAP at 250 µg / ml (for asd knockouts). Screening for knockout mutants was then done through PCR, and the correct knockouts were selected, re-streaked, and confirmed for the loss of the conjugated plasmid through DNA sequencing and its inability to grow in presence of kanamycin and its auxotrophy for the respective metabolite (d-glutamate or DAP). To cure the knockouts from the ampicillin resistance plasmid, they were cultured and 40 °C for the loss of ampicillin resistance. To knockout murI in S. enterica VPN20009, we used the same process except that we had to transform it with the p101-Amp-murI plasmid containing an extra copy of the murI gene before knocking out the chromosomal murIgene. After knocking out the chromosomal murI gene, VNP20009 ΔmurI mutant was cured from plasmid p101-Amp-murI through culturing on LB plates supplemented with d-glutamate in absence of ampicillin at 40 °C and screening for the loss of ampicillin resistance. To construct S. enterica VNP20009m strain, murI gene was supplemented in trans under the constitutive promoter PJ23113 on a plasmid (pMurI) harboring also chloramphenicol resistance cassette and pBR322 origin. Attorney Docket Number: CCF-42779.601 Mass spectrometry analyses for kynurenine in mice tissues and cell cultures Mice tissues were minced with scalpel, then 50~200 mg portions of the minced tissues were mixed with 3 volumes distilled water in 2 ml Eppendorf tubes. The suspensions were first heated at 95 °C to deactivate the enzymes in the kynurenine pathway then homogenized using a bead tissue homogenizer (MM400, Retsch). All samples were then filtered through a 3 KDa cut-off membrane filter (Amicon®, UFC5003BK) to remove the proteins and high molecular weight components. The samples (25 ul) were mixed with 2.5 µl of internal standard solution containing 100 µM of each of [2H4]-L-kynurenine (Cat# D-8026, CDN isotopes), and [2H5]-L-tryptophan (Cat# D-1522, CDN isotopes) and were then injected onto LC / MS-MS for quantitation.2 µl of the prepared samples were injected onto LC column through Shimadzu autosampler (SIL-HTc) and metabolites were resolved on C18 column (Prodigy, 150 x 2 mm, 5 micron, 00F-3300-B0, Phenomenx) with LC gradients generated from binary pumps (Shimadzu LC-20AD) connected to two solvents, A: 0.2% formic acid in water; B: 0.2% formic acid in methanol. The LC elutes were analyzed on API 5000 Mass spectrometer (Sciex) with an electrospray ion source. Standards and internal standards were monitored in positive MRM mode with parent to daughter transitions: m / z 209→94for kynurenine, m / z 205→188 for tryptophan, m / z 213→96 for [2H4] -L-kynurenine and m / z 210→192 for [2H5] tryptophan. MS parameters were optimized for individual standards. Standard curves were generated from serial dilutions of standards undergoing the same procedures as biological samples. For some of the earlier experiments, [¹³C₁₀]-L-kynurenine (Cat# CLM-9884, Campridge Isotope Laboratories) was used as internal standard instead of [D4]-L-kynurnenine and the parent to daughter ion transition for monitoring [¹³C₁₀]-L- kynurenine was m / z 219→100. For experiments assessing the concentration of kynurenic acid, [2H5]- kynurenic acid (Cat# D-439, CDN isotopes) was used as internal standard. The parent to daughter ion transitions for monitoring kynurenic acid and [2H5]- kynurenic acid were m / z 189→144 and m / z 194→166, respectively. Cell Lines and Cell Culture All cell lines used in this study are summarized in Table 3. Mouse ovarian epithelial cancer cell line ID8 (50), KPCA.A (39), and BPPNM (39) (All are C57Bl / 6 mouse strain syngeneic) were cultured in Dulbecco Modified Eagle Medium (DMEM) media containing 5% heat-inactivated fetal bovine serum (FBS, Atlas Biologicals Cat # F-0500-D), 1% Insulin-Transferrin-Selenium (Thermo Fisher Scientific; ITS-G, 41400045), 100μl EGF (10ug / ml), and grown under standard conditions (Incubation at 37 °C and 5% CO2). KPCA.A Attorney Docket Number: CCF-42779.601 and BBPNM cell lines were a kind gift from Dr. Robert A. Weinberg at Whitehead Institute for Biomedical Research to the Reizes lab (39). The mouse triple negative breast cancer cell line 4T1-Luc (Luciferase-expressing 4T1 cells, BALB / c syngeneic) was a gift from Dr. William Schiemann at Case Western Reserve University.4T1-luc cells were routinely cultured in either RPMI media or DMEM media containing 10% heat-inactivated FBS. Construction of 4T1-EV and 4T1-IDO1 breast cancer cell lines Lentiviral vector harboring the murine ido1 gene together with a fusion Luciferase- tdTomato protein (IDO1 vector) was constructed by VectorBuilder. A control empty vector that lacks ido1 gene (EV) was also constructed. Both vectors were purified from E. coli using Plasmid Maxiprep kit (Nucleospin). Viral particle preparations were carried out in HEK293T cells cells following a previously described method (77). In brief, HEK293T cells were cultured in a 100 mm dish. When reaching 60% confluency, HEK293T cells were transfected with pMD2.G (Lentiviral envelope-expressing plasmid), pRSV-Rev (Lentiviral packaging plasmid), pMDLg / Prre (Packaging plasmid containing Gag and Pol), and either the Empty vector (EV) or the Ido1-expressing vector (IDO1 vector) using Lipofectamine 3000 reagents (Thermo Fischer Scientific). After 24 hours, the transfection medium was removed, and fresh serum-enriched DMEM media was added to the HEK293T cells. Following an additional 24 hours, DMEM media containing lentiviral particles were filtered, and the filtered media was added to 4T1-Luc cells. After 3 days, the media was changed, and regular DMEM-10% FBS media was added to the 4T1-Luc cells. Transduced cells were cultured, and flow cytometry was performed to isolate tdTomato-positive 4T1-Luc cells (Top 10%). The sorted cells were cultured, stored as aliquots, and utilized for further experiments. Testing S. enterica response to kynurenine in cancer cell culture supernatants Cancer cell lines were cultured in 1 ml of the 5%serum supplemented DMEM media within 6-well plates at ~ 100,000 cells per well. IFNγ (Peprotech, Cat#315-05) was added to the test wells at 200 ng / ml concentration. The plates were incubated for 72 h under standard incubation conditions (37 °C, 5% CO2). The supernatants were then collected, centrifuged and 200 µl aliquots were processed for LC-MS / MS analyses.100 µl samples from the supernatants were then aliquoted in 96 well plates and used as culturing media for the engineered S. enterica. Primary overnight cultures of the engineered S. enterica harboring pKynR5-mCherry dual plasmid system (or the control pKynR5-nLuc) were prepared in LB media, washed, and then 2 µl of the bacterial suspensions were spotted in the wells Attorney Docket Number: CCF-42779.601 containing the spent cancer cells supernatants. The plates were incubated within Tecan Pro infinite microplate reader with shaking at 37 °C and the OD600and mCherry fluorescence were measured. Data shown in Figure 3 are at 9 h time point. Testing S. enterica in murine 4T1 and KPCA.A tumor models All experiments involving mice were performed using protocols approved by the Cleveland Clinic Animal Care and Use Committee (IACUC protocols 2706, 3279, and 2924). For the mice experiments, murI gene in the control S. enterica VPN20009 strain was knocked out and supplemented in trans under the constitutive promoter PJ23113 on a plasmid (pMurI) harboring also chloramphenicol resistance cassette and pBR322 origin. This was done to ensure the absence of contaminating colonies upon counting the bacteria from mice tissues on agar plates which were supplemented with chloramphenicol. For KPCA.A tumor models, cells were injected subcutaneously in the flank region in C57BL / 6J mice. For 4T1 tumor models, cells were injected either in the flank or in the 3rdmammary fat pads in BALB / c mice. For experiments using 4T1-IDO1 and 4T1-EV cell lines, they were injected subcutaneously on opposite flanks in immunocompromised NSG mice38. S. entrica strains were grown overnight in LB media supplemented with DAP, d- glutamate and chloramphenicol. Kanamycin was also added to AD95+ cultures. At the day of the bacterial injection, bacterial cultures were washed and resuspended in PBS at an OD600 of 0.1.100 µl of these suspensions were then injected in the mice intraperitoneally, intravenously (through retroorbital route) or through direct intratumoral injection. Unless mentioned otherwise, the bacterial dose was equivalent to around 2~4x106cfu based on colony counting on agar plates. Mice were anesthetized through isoflurane inhalation anesthesia during the injection and were monitored daily after the bacterial injection. For retroorbital injection, a drop of ophthalmic anesthetic (0.5 % proparacaine hydrochloride ophthalmic solution) was placed on the eye before injection. This provided additional procedural and post-procedural analgesia. At the specified days after bacterial injection, mice were euthanized through carbon dioxide asphyxia, and organs were extracted immediately after euthanasia. Each organ was cut into small pieces using scalpel, then divided into two portions. One portion was snap frozen for later processing for LC-MS / MS analyses while the other portion was weighed, suspended in 1x PBS and homogenized using bead homogenizer, diluted, and plated on LB agar plates supplemented with d-glutamate, DAP, and chloramphenicol for colony counting. For statistical analyses, mice which showed < 5 cfu / mg in the tumor samples were considered outliers and were excluded. Attorney Docket Number: CCF-42779.601 Rigor and statistical analysis Each key in vitro experiment was repeated at least twice. For statistical analysis, we used one-way ANOVA to compare different groups followed by Tukey Post hoc test when the samples are normally distributed. Kruskal-Wallis (for un-matched groups) and Friedman tests (for matched groups) were used for statistical analyses of data which are not normally distributed. Likewise, student t-test, and Mann-Whitney U test were used for pair-wise comparisons. Analysis was performed using GraphPad Prism 10. For animal experiments and based on differences in means we consider to be biologically relevant, and typical standard deviations seen in such data in previous studies, number of animals needed for each group were estimated so that we can detect statistical significance at P <0.05. We used only female mice as our studies are focusing on breast and ovarian cancers which affect mainly female patients. Mice were assigned randomly to different groups. Laboratory personnel were not blinded during the animal experiments to avoid cross-contamination between groups. Each point on the graphs represents an individual mouse. REFERENCES: 1. Chein, T., Doshi, A. & Danino, T. Advances in bacterial cancer therapies using synthetic biology. Current opinion in systems biology 5, 8 (2017). 2. Daschner, P.J., Rasooly, A. & White, J.D. Bugs as Cancer Drugs: Challenges and Opportunities. Mol Cell Biol 39(2019). 3. Salicrup, L.A., Ossandon, M., Prickril, B. & Rasooly, A. Bugs as Drugs, potential self-regenerated innovative cancer therapeutics approach for global health. J Glob Health 10, 010311 (2020). 4. Duong, M.T., Qin, Y., You, S.H. & Min, J.J. Bacteria-cancer interactions: bacteria- based cancer therapy. Exp Mol Med 51, 1-15 (2019). 5. Pawelek, J.M., Low, K.B. & Bermudes, D. Tumor-targeted Salmonella as a novel anticancer vector. Cancer Res 57, 4537-4544 (1997). 6. Low, K.B., et al. Lipid A mutant Salmonella with suppressed virulence and TNFalpha induction retain tumor-targeting in vivo. Nat Biotechnol 17, 37-41 (1999). 7. Danino, T., Lo, J., Prindle, A., Hasty, J. & Bhatia, S.N. In Vivo Gene Expression Dynamics of Tumor-Targeted Bacteria. Acs Synth Biol 1, 465-470 (2012). 8. Rosenberg, S.A., Spiess, P.J. & Kleiner, D.E. Antitumor effects in mice of the intravenous injection of attenuated Salmonella typhimurium. J Immunother 25, 218-225 (2002). Attorney Docket Number: CCF-42779.601 9. Toso, J.F., et al. Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma. J Clin Oncol 20, 142-152 (2002). 10. Amobi-McCloud, A., et al. IDO1 Expression in Ovarian Cancer Induces PD-1 in T Cells via Aryl Hydrocarbon Receptor Activation. Frontiers in immunology 12, 678999 (2021). 11. de Jong, R.A., et al. Serum tryptophan and kynurenine concentrations as parameters for indoleamine 2,3-dioxygenase activity in patients with endometrial, ovarian, and vulvar cancer. Int J Gynecol Cancer 21, 1320-1327 (2011). 12. Smith, L.P., Bitler, B.G., Richer, J.K. & Christenson, J.L. Tryptophan catabolism in epithelial ovarian carcinoma. Trends Cancer Res 14, 1-9 (2019). 13. Zimmer, P., et al. Resistance Exercise Reduces Kynurenine Pathway Metabolites in Breast Cancer Patients Undergoing Radiotherapy. Front Oncol 9, 962 (2019). 14. Sakurai, K., et al. [Study of indoleamine 2,3-dioxygenase expression in patients with breast cancer]. Gan To Kagaku Ryoho 32, 1546-1549 (2005). 15. Heng, B., et al. Differential kynurenine pathway metabolism in highly metastatic aggressive breast cancer subtypes: beyond IDO1-induced immunosuppression. Breast Cancer Res 22, 113 (2020). 16. Heng, B., et al. Understanding the role of the kynurenine pathway in human breast cancer immunobiology. Oncotarget 7, 6506-6520 (2016). 17. Venkateswaran, N., et al. MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer. Genes Dev 33, 1236-1251 (2019). 18. Crotti, S., et al. Tryptophan Catabolism and Response to Therapy in Locally Advanced Rectal Cancer (LARC) Patients. Front Oncol 10, 583228 (2020). 19. Sun, X.Z., et al. Alteration of fecal tryptophan metabolism correlates with shifted microbiota and may be involved in pathogenesis of colorectal cancer. World journal of gastroenterology : WJG 26, 7173-7190 (2020). 20. Lin, D.J., et al. The immunotherapeutic role of indoleamine 2,3-dioxygenase in head and neck squamous cell carcinoma: A systematic review. Clinical otolaryngology : official journal of ENT-UK ; official journal of Netherlands Society for Oto-Rhino-Laryngology & Cervico-Facial Surgery 46, 919-934 (2021). 21. Uyttenhove, C., et al. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 9, 1269-1274 (2003). 22. Puccetti, P., et al. Accumulation of an Endogenous Tryptophan-Derived Metabolite in Colorectal and Breast Cancers. PloS one 10(2015). Attorney Docket Number: CCF-42779.601 23. Nguyen, N.T., et al. Aryl hydrocarbon receptor and kynurenine: recent advances in autoimmune disease research. Frontiers in immunology 5, 551 (2014). 24. D'Amato, N.C., et al. A TDO2-AhR signaling axis facilitates anoikis resistance and metastasis in triple-negative breast cancer. Cancer Res 75, 4651-4664 (2015). 25. Ye, Z., Yue, L., Shi, J., Shao, M. & Wu, T. Role of IDO and TDO in Cancers and Related Diseases and the Therapeutic Implications. J Cancer 10, 2771-2782 (2019). 26. Aslakson, C.J. & Miller, F.R. Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer Res 52, 1399-1405 (1992). 27. Schrors, B., et al. Multi-Omics Characterization of the 4T1 Murine Mammary Gland Tumor Model. Front Oncol 10, 1195 (2020). 28. Iyer, S., et al. Genetically Defined Syngeneic Mouse Models of Ovarian Cancer as Tools for the Discovery of Combination Immunotherapy. Cancer Discov 11, 384-407 (2021). 29. Zuo, H., et al. Plasma Biomarkers of Inflammation, the Kynurenine Pathway, and Risks of All-Cause, Cancer, and Cardiovascular Disease Mortality: The Hordaland Health Study. Am J Epidemiol 183, 249-258 (2016). 30. Doublet, P., van Heijenoort, J., Bohin, J.P. & Mengin-Lecreulx, D. The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity. J Bacteriol 175, 2970-2979 (1993). 31. Han, Q., Fang, J. & Li, J. Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase. Biochem J 360, 617-623 (2001). 32. Jansen, R.S., et al. Aspartate aminotransferase Rv3722c governs aspartate-dependent nitrogen metabolism in Mycobacterium tuberculosis. Nat Commun 11, 1960 (2020). 33. Jawalagatti, V., Kirthika, P. & Lee, J.H. Targeting primary and metastatic tumor growth in an aggressive breast cancer by engineered tryptophan auxotrophic Salmonella Typhimurium. Mol Ther Oncolytics 25, 350-363 (2022). 34. Lu, Q., et al. A host-adapted auxotrophic gut symbiont induces mucosal immunodeficiency. Science 385, eadk2536 (2024). 35. Zhao, M., et al. Tumor-targeting bacterial therapy with amino acid auxotrophs of GFP-expressing Salmonella typhimurium. Proc Natl Acad Sci U S A 102, 755-760 (2005). 36. Low, K.B., et al. Construction of VNP20009: a novel, genetically stable antibiotic- sensitive strain of tumor-targeting Salmonella for parenteral administration in humans. Methods Mol Med 90, 47-60 (2004). Attorney Docket Number: CCF-42779.601 37. Clairmont, C., et al. Biodistribution and genetic stability of the novel antitumor agent VNP20009, a genetically modified strain of Salmonella typhimurium. J Infect Dis 181, 1996- 2002 (2000). 38. Shultz, L.D., et al. Human lymphoid and myeloid cell development in NOD / LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells. J Immunol 174, 6477-6489 (2005). 39. Staedtke, V., Roberts, N.J., Bai, R.Y. & Zhou, S. Clostridium novyi-NT in cancer therapy. Genes Dis 3, 144-152 (2016). 40. Staedtke, V., et al. Neutrophil depletion enhanced the Clostridium novyi-NT therapy in mouse and rabbit tumor models. Neurooncol Adv 4, vdab184 (2022). 41. Munn, D.H. & Mellor, A.L. IDO in the Tumor Microenvironment: Inflammation, Counter-Regulation, and Tolerance. Trends Immunol 37, 193-207 (2016). 42. Ait-Belkacem, R., et al. Microenvironment Tumor Metabolic Interactions Highlighted by qMSI: Application to the Tryptophan-Kynurenine Pathway in Immuno-Oncology. SLAS Discov 22, 1182-1192 (2017). 43. Lee, K.C., Zheng, L.M., Margitich, D., Almassian, B. & King, I. Evaluation of the acute and subchronic toxic effects in mice, rats, and monkeys of the genetically engineered and Escherichia coli cytosine deaminase gene-incorporated Salmonella strain, TAPET-CD, being developed as an antitumor agent. Int J Toxicol 20, 207-217 (2001). 44. King, I., et al. Tumor-targeted Salmonella expressing cytosine deaminase as an anticancer agent. Hum Gene Ther 13, 1225-1233 (2002). 45. Sasaki, T., et al. Genetically engineered Bifidobacterium longum for tumor-targeting enzyme-prodrug therapy of autochthonous mammary tumors in rats. Cancer Sci 97, 649-657 (2006). 46. Gniadek, T.J., et al. A Phase I, Dose Escalation, Single Dose Trial of Oral Attenuated Salmonella typhimurium Containing Human IL-2 in Patients With Metastatic Gastrointestinal Cancers. J Immunother 43, 217-221 (2020). 47. Gurbatri, C.R., et al. Engineered probiotics for local tumor delivery of checkpoint blockade nanobodies. Sci Transl Med 12(2020). 48. Chowdhury, S., et al. Programmable bacteria induce durable tumor regression and systemic antitumor immunity. Nat Med 25, 1057-1063 (2019). 49. Santhanam, S., Alvarado, D.M. & Ciorba, M.A. Therapeutic targeting of inflammation and tryptophan metabolism in colon and gastrointestinal cancer. Transl Res 167, 67-79 (2016). Attorney Docket Number: CCF-42779.601 50. Chen, W., et al. Bacteria-Driven Hypoxia Targeting for Combined Biotherapy and Photothermal Therapy. ACS Nano 12, 5995-6005 (2018). 51. Yu, B., et al. Explicit hypoxia targeting with tumor suppression by creating an "obligate" anaerobic Salmonella Typhimurium strain. Scientific reports 2, 436 (2012). 52. Chien, T., et al. Enhancing the tropism of bacteria via genetically programmed biosensors. Nat Biomed Eng (2021). 53. Datsenko, K.A. & Wanner, B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci U S A 97, 6640-6645 (2000). All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in chemistry, medicine, and molecular biology or related fields are intended to be within the scope of the following claims.

Claims

Attorney Docket Number: CCF-42779.601 CLAIMS: We claim:

1. A composition comprising: a bacterial cell, wherein said bacterial cell comprises one or more nucleic acid sequences, wherein said one or more nucleic acid sequences comprise: i) a first nucleotide sequence encoding a kynurenine transcriptional regulator protein, ii) a second nucleotide sequence encoding a kynurenine transporter protein, and iii) optionally a third nucleotide sequence comprising a kynurenine responsive promoter upstream of a plasmid replication gene encoding a plasmid replication protein; and iv) optionally a fourth nucleotide sequence comprising a kynurenine responsive promoter upstream of a reporter gene encoding a reporter protein; and v) optionally a fifth nucleotide sequence comprising a kynurenine responsive promoter upstream of a gene important or essential for growth of the bacteria or a gene codes for therapeutic payload.

2. The composition of claim 1, wherein said third and / or fourth nucleotide and / or fifth nucleotide sequence is present in said one or more nucleic acid sequences.

3. The composition of claim 1, wherein said one or more nucleic acid sequences comprises at least one plasmid sequence.

4. The composition of claim 1, wherein said one or more nucleic acid sequences comprises first and second plasmid sequences, or are part of the chromosome of said bacteria.

5. The composition of claim 4, wherein said first plasmid sequence contains said first and second nucleotide sequences, and wherein said second plasmid sequence contains said third and / or fourth nucleotide sequences.

6. The composition of claim 1, wherein said kynurenine transcriptional regulator protein comprises KynR protein from Pseudomonas aeruginosa or a KynR protein from a Gram- negative and Gram-positive bacteria.Attorney Docket Number: CCF-42779.601 7. The composition of claim 1, wherein said kynurenine transporter protein comprises an Mtr protein, Tna protein, AroP protein, TyrP, or PheP.

8. The composition of claim 1, wherein said kynurenine responsive promoter comprises a kynurenine-responsive promoter DNA sequence (Pkyn).

9. The composition of claim 1, wherein said plasmid replication protein comprises repL.

10. The composition of claim 1, wherein said reporter protein is selected from mCherry, luciferase, GFP, tdTomato, CFP, RFP, YFP, β-galactosidase and β-galactosidase.

11. The composition of claim 1, wherein said bacteria is from the following genera which naturally harbor KynR selected from: Salmonella, Escherichia, Shigella, Proteus, Enterobacteriaceae, Pseudomonas, Cupriavidus, other Gamma proteobacteria, and Bacillus.

12. The composition of claim 1, wherein said bacteria is E. coli.

13. The composition of claim 1, wherein said therapeutic is selected from: immunomodulatory agents, bacterial toxins, and drug-activating enzymes.

14. A method comprising: administering the bacteria of any one of claims 1-13 to a patient with a tumor or a disease in which kynurenine is accumulating in localized lesions, optionally wherein said patient is a human.

15. The method of claim 14, wherein said administration is such that said bacteria localizes to said tumor, or said localized lesions, of said patient.

16. The method of claim 14, wherein said tumor or a localized lesion produces kynurenine and said bacteria preferentially localizes, and / or proliferates in, said tumor or lesion at least in part based on said kynurenine expression.

17. The method of claim 14, wherein these bacteria are administered orally, systemically or through direct intratumor, or intra-lesion, injection.Attorney Docket Number: CCF-42779.601 18. A kit or system comprising: a) said bacteria of any one of claims 1-13; and b) a delivery device or composition for administering said bacteria to a patient with a tumor, optionally wherein said patient is a human.

19. The kit or system of claim 18, wherein said delivery device or composition is selected from: syringes, oral tablets, capsules, enema, and IV solution.

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