Treatment of nervous system tumors using attenuated salmonella typhimurium

Abstract

Provided herein are compositions and methods for the treatment of benign nervous system tumors, including neurosheathing tumors, using attenuated mutants of Salmonella typhimurium comprising a spiC deletion and optionally one or more checkpoint inhibitors.

Description

[0001] Priority Statement

[0002] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 453,202, filed March 20, 2023. The entire foregoing is incorporated herein by reference. Technical Field

[0003] This article provides compositions and methods for treating benign nervous system tumors, including schwannoma, using an attenuated mutant of Salmonella Typhimurium containing spiC deletion and optionally one or more checkpoint inhibitors. Background Technology

[0004] Neurofibromas are slow-growing, benign neoplasms originating from Schwann-lineage cells. 1,2 Depending on their location and size, these tumors can cause a wide range of neurological deficits, including hearing loss, imbalance, tinnitus, motor impairment, and severe pain. 3,4 In some cases, they can lead to death due to brainstem compression. 5 Schwannomas can occur sporadically (hence the term "sporadic schwannomas") or as part of the debilitating genetic syndromes NF2-associated schwannomatosis (NF2 or NF2-SWN) and schwannomatosis (SWN). 6 Treatment for schwannomas is primarily limited to surgical resection and symptom management of pain. Resection is often non-curable for many patients, frequently associated with additional nerve damage, and may be impractical due to the location or large number of tumors. 7 Due to the slow-replicating nature of these benign lesions, the efficacy of anticancer therapies for schwannomas has not yet been proven. 8-10 Bevacizumab is currently the only universally accepted drug therapy for schwannomas; it temporarily stabilizes tumor growth by targeting the highly vascularized nature of these neoplasm subpopulations. 8,11 Unfortunately, current pain management strategies are insufficient for many people, further increasing the burden of the disease. The fact that schwannomas can occur in multiple locations and develop new lesions throughout a person's life further complicates treatment. Therefore, schwannomas and related diseases cause lifelong suffering that cannot be stably controlled with current treatment options.

[0005] In summary, schwannomas are slow-growing benign growths that develop throughout the body, including along the spinal cord and intracranially, associated with peripheral nerves. Schwannomas often first appear in childhood or adolescence and can develop new tumors throughout life. These tumors cause pain, sensory / motor dysfunction, and death by compressing peripheral nerves, the spinal cord, and / or the brain. The immense suffering and debilitating effects associated with schwannomas, coupled with the lack of treatment options, make their treatment a major unmet medical need. Summary of the Invention

[0006] This article describes a treatment approach for benign growths, including schwannomas, involving intratumoral (it) injection of one of several attenuated deletion mutants of Salmonella Typhimurium.

[0007] The term 'deletion mutant' hereinafter refers to a strain genetically engineered to lack the specific chromosomal DNA sequence required to encode a specific protein with a known function. The described *Salmonella typhimurium* deletion mutants include strains with a deletion in a gene called *spiC*. *SpiC* encodes a key component of the type 3 secretion system (T3SS) of *Salmonella typhimurium*. The T3SS of *Salmonella typhimurium* is a nanomachine used to allow the bacterium to enter the cytosol of host mammalian cells, then kill the host cells or inhibit their function. These cells include macrophages and other immune cells important for tumor elimination. The *spiC* mutant, referred to as the *spiC* F6-Y129 deletion mutant (or VNP-△spiC or simply △spiC in the figures), is the focus of this invention. The △spiC strain was found to be highly attenuated in virulence compared to its parent strain VNP20009 (also indicated as YS1646), and was also found to be equivalent in potency to its parent strain against schwannomas in a mouse model of NF2 disease.

[0008] Data support intratumoral (it) injection of a live attenuated *Salmonella typhimurium* spiCF6-Y129 deletion mutant, optionally in combination with PD-1 checkpoint inhibitors, as an immunotherapy capable of controlling the growth of both injected and non-injected benign nervous system tumors, including schwannomas and neurofibromatosis-associated vegetations, including neurofibromatosis type 1 (NF1)-associated tumors and meningiomas. Data further demonstrate the potential of this treatment strategy to control tumor growth following initial treatment. Importantly, direct injection of VNP20009 (the parental strain of *Salmonella typhimurium* spiCF6-Y129 deletion mutant) into tumors exhibits a vaccine-like effect in inducing a systemic antitumor immune response. 12The spiC F6-Y129 deletion mutant of VNP20009 showed similar potency to VNP20009, while also exhibiting reduced virulence in an acute lethal model of systemic disease in mice caused by Salmonella typhimurium (Figure 1).

[0009] Therefore, this article provides a method for treating subjects with benign nervous system tumors or at risk of developing benign nervous system tumors (subjects at risk include those with conditions associated with the development of these tumors, including neurofibromatosis type 1 (NF1); NF2-associated schwannomatosis (NF2 or NF2-SWN); schwannomatosis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF)). The method comprises administering to the subject a therapeutically effective amount of a composition comprising a live attenuated *Salmonella typhimurium* spiCF6-Y129 deletion mutant, optionally in combination with an immune checkpoint inhibitor and / or angiogenesis inhibitor. This article also provides a method for treating subjects with benign nervous system tumors or at risk of developing benign nervous system tumors using a composition comprising a live attenuated *Salmonella typhimurium* spiCF6-Y129 deletion mutant, optionally in combination with a checkpoint inhibitor and / or angiogenesis inhibitor.

[0010] In some implementations, the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the following groups: neurofibromatosis type 1 (NF1); NF2-associated schwannoma (NF2 or NF2-SWN); schwannoma; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof. In some implementations, the subject does not have a malignant solid tumor (i.e., has not been diagnosed with a malignant solid tumor). In some implementations, the subject has a condition associated with an increased risk of benign nervous system tumors, such as neurofibromatosis type 1 (NF1); NF2-associated schwannoma (NF2); or schwannoma.

[0011] In some implementations, the attenuated spiC F6-Y129 deletion mutant of Salmonella Typhimurium VNP20009 is administered intratumorally or intravenously. The key implementation of the spiC F6-Y129 mutation is the fact that it is an in-frame deletion of the spiC gene. This deletion is defined as the loss of the codons encoding the spiC gene from phenylalanine 6 to tyrosine 129. Therefore, the spiC F6-Y129 deletion mutation removes the coding sequence for the SpiC protein between phenylalanine 6 and tyrosine 129. This spiC F6-Y129 deletion construct will be referred to hereinafter as spiC F6-Y129 deletion or simply △spiC. Mutant derivatives of all parental strains carrying this genetic alteration in their genome will be referred to as spiCF6-Y129 deletion mutants or simply △spiC.

[0012] In some implementations, the attenuated spiC F6-Y129 deletion mutant includes derivatives of the Salmonella enterica serovar Typhimurium strain VNP20009. 13-16 The genomic sequence of VNP20009 defines mutations in genes that alter lipid A acylation (e.g., in the msbB gene) or block purine biosynthesis (e.g., in the pull gene). 13 Derivatives of VNP20009 carrying the deletion of spiC F6-Y129 are also referred to herein as VNP-△spiC or simply as △spiC in the accompanying drawings of this application.

[0013] In some implementations, the checkpoint inhibitor is an inhibitor of PD-1 signaling or CTLA-4 signaling, such as an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

[0014] In some implementations, the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR), such as bevacizumab.

[0015] Furthermore, this document provides a method for treating benign nerve sheath tumors in mammals, comprising administering to the mammal a therapeutically effective dose or titer of an attenuated strain of pathogenic intestinal bacteria. In some embodiments, the attenuated strain comprises a spiC F6-Y129 deletion mutant of Salmonella Typhimurium VNP20009.

[0016] This article provides a method for treating a subject with or at risk of developing a nervous system tumor, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a live attenuated spiC-deficient strain of Salmonella bacteria, optionally in combination with an immune checkpoint inhibitor and / or angiogenesis inhibitor.

[0017] In addition, this article provides a composition comprising a live attenuated spiC-deficient variant of Salmonella bacteria, optionally in combination with a checkpoint inhibitor and / or angiogenesis inhibitor, for use in treating subjects with benign nervous system tumors or at risk of developing benign nervous system tumors.

[0018] In some implementations, the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the following groups: neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannoma; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof. In some implementations, the subject does not have a malignant solid tumor. In some implementations, the subject has a condition associated with an increased risk of benign nervous system tumors. In some implementations, the condition associated with an increased risk of benign nervous system tumors is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannoma.

[0019] In some implementations, attenuated Salmonella is administered intratumorally or intravenously.

[0020] In some implementations, the attenuated spiC-deleted variant of Salmonella is an attenuated strain of Salmonella typhimurium.

[0021] In some implementations, the attenuated spiC-deficient variant of Salmonella typhimurium is the enteric Salmonella typhimurium serotype VNP20009.

[0022] In some embodiments, the composition does not contain Clostridium novyi.

[0023] In some implementations, the attenuated Salmonella species does not contain a cleavage gene or cassette that is operatively linked to an intracellularly induced Salmonella promoter.

[0024] In some implementations, the attenuated Salmonella strain is not VNP20009.

[0025] In some implementations, the checkpoint inhibitor is an inhibitor of PD-1 signaling or CTLA-4 signaling.

[0026] In some implementations, the inhibitor of PD-1 signaling is an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

[0027] In some embodiments, the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR). In some embodiments, the VEGF inhibitor is bevacizumab.

[0028] This article also provides compositions comprising a live attenuated spiC deletion variant of enteric Salmonella typhimurium serotype VNP20009 (e.g., as described herein).

[0029] In some embodiments, administration includes, but is not limited to, intravenous injection or direct injection into a benign schwannoma. In some embodiments, the schwannoma includes, but is not limited to, neurofibroma or schwannoma. In some embodiments, the tumor includes, but is not limited to, those associated with neurofibromatosis type 1, NF2-associated schwannoma, schwannoma disease, or sporadic schwannoma. In some embodiments, the method includes administering to the mammal a therapeutically effective dose of an attenuated splice variant of pathogenic intestinal bacteria and a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor includes, but is not limited to, peptides, antibodies, small molecules, microRNAs, antisense oligonucleotides, or small interfering RNAs. In some embodiments, the checkpoint inhibitor is a monoclonal antibody that binds to an epitope of an antigen. In some embodiments, the monoclonal antibody binds to an epitope in the PD-1 or CTFA-4 antigen. In some embodiments, the mammal is a human.

[0030] This document also provides pharmaceutical compositions comprising an attenuated VNP-ΔspiC in a pharmaceutically acceptable carrier and optionally a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is a monoclonal antibody. In some embodiments, the monoclonal antibody binds to an epitope of the PD-1 or CTFA-4 antigen. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. These materials, methods, and examples are illustrative only and are not intended to be limiting.

[0031] All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Other features and advantages of the invention will become apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0032] Figure 1A -F. Evaluation of tumor control by VNP20009 SPI-1 and SPI-2 mutant strains in a mouse model of multiple tumors. In an NF-2 syngeneic mouse model, intratumoral injection of SPI-1 ΔsipB (A), ΔspiC (B), and VNP20009 mutants was performed. Tumor volume was monitored over time (08031) in FVB / N mice and compared with PBS-treated mice (n=8 / group). Two-way ANOVA with repeated measures and Tukey's multiple comparison test were used to compare samples. Data are presented as mean + / - SEM. C. Biodistribution of VNP20009, ΔspiC, and spiC overexpressing strains on days 2, 7, and 12 after intratumoral injection. Bacterial colonization was determined by plating excised and sonicated tissue tumors from mouse NF2 syngeneic experiments onto LB plates and calculated as CFU / g (n=3 / group). Samples at different time points were compared using one-way ANOVA and Dunnett's multiple comparison test. Data are presented as mean + / - SEM. Intratumoral injection of ΔspiC and VNP20009 mutant strains was performed in syngeneic (D) and xenograft (F) schwannomas. Tumor volume was monitored over time in FVB / N (D) and nude (F) mice treated with PBS and compared with PBS-treated mice (n=8 / group). Tumor signal was compared using repeated measures two-way ANOVA and Tukey's test. Data are presented as mean + / - SEM. E. Biodistribution of VNP20009 and ΔspiC strains on day 22 after intratumoral injection. Bacterial colonization was determined by plating excised and sonicated tissue tumors from syngeneic schwannomas in LB plates and calculated as CFU / g (n=5 / group). The Mann-Whitney test was used to compare samples. The data is shown as the average value + / - SEM.

[0033] Figure 2A -D. Compared to the VNP20009 wt strain, the ΔspiC mutant showed less toxicity in FVB / N mice. A. Intraperitoneal (ip) injection of PBS, 2x10 7Body weight was monitored for 3 days in the case of VNP20009 wt or ΔspiC mutant strains with CFU. Data were presented as percentage weight loss (n=5 mice / group). Samples were compared using two-way ANOVA with repeated measures and Tukey's multiple comparison test. Data were presented as mean + / - SEM. B. Biodistribution of VNP20009 and ΔspiC strains in the liver and spleen on day 3 after intraperitoneal injection. Bacterial colonization was determined by plating excised and sonicated liver and spleen tissues in LB plates and calculated as CFU / g (n=5 / group). Samples were compared using a paired t-test. Data were presented as mean + / - SEM. C. Liver and spleen tissues from mice injected with VNP2009 and ΔspiC were weighed on day 3 after intraperitoneal injection and compared with control mice injected with PBS. The figure shows the weight of the tissues (n=5 mice / group). Samples were compared using a standard one-way ANOVA and Tukey's multiple comparison test. The data is shown as the average value + / - SEM.

[0034] Figure 3A -D. Compared with the VNP20009 wt strain, the ΔspiC mutant showed less toxicity in FVB / N mice. A. Time-dependent survival of mice injected with VNP2009, ΔspiC, and PBS. Long-rank (Mantel-Cox) test was used to compare samples (n=5 mice / group). B. Intraperitoneal injection of PBS, 2x10 6 Body weight was monitored over 10 days in the case of VNP20009 wt and ΔspiC mutant strains of CFU. Data were presented as percentage weight loss (n=5 mice / group). Mice injected with VNP20009 and ΔspiC were compared with PBS controls using repeated measures two-way ANOVA and Tukey's multiple comparison test. Data were presented as mean + / - SEM. C. Intraperitoneal injection of 2x10 6 Biodistribution of VNP20009 and ΔspiC strains in the liver and spleen on day 10 after CFU administration. Bacterial colonization was determined by platenting resected and sonicated liver and spleen tissues in LB plates and calculated as CFU / g (n=5 / group). One-way ANOVA and Tukey's multiple comparison test were used to compare samples. Data are presented as mean + / - SEM. D. Liver (left) and spleen (right) tissues from mice injected with VNP2009 and ΔspiC were weighed on day 3 after intraperitoneal injection and compared with control mice injected with PBS. The figure shows the weight of the tissues (n=5 mice / group). Plain one-way ANOVA and Tukey's multiple comparison test were used to compare samples. Data are presented as mean + / - SEM.

[0035] Figure 4A -B. Deletion of the VNP20009 gene in Salmonella Typhimurium SPI-1 and SPI-2 did not impair growth in standard laboratory media or invasion in relevant cell lines. A. Growth of VNP20009 and mutant derivatives was monitored over time by measuring optical density and plotted on an algorithmic scale (Log10). B. Data are presented as the mean of three technical replicates + / - SD. HEI-193, Caco-2, and RAW 264.7 cells were infected at 37°C for 1 hour with parental strain 14028, VNP20009 wt, and mutant strain at MOIs of 50, 10, and 5, respectively, followed by gentamicin treatment. The number of invasive bacteria was determined by recording CFU / ml / T0. Results are expressed as the percentage of invasion (CFU / ml / T0). Each infection is presented as the mean of at least three independent experiments + / - SEM. The samples were compared with the VNP20009 control using ordinary one-way ANOVA and Dunnett's multiple comparison test.

[0036] Figure 5 Overexpression of the VNP20009 gene in *Salmonella typhimurium* SPI-1 and SPI-2 did not increase invasion of Caco-2 and RAW 264.7 cells. Caco-2 and RAW 264.7 cells were infected at 37°C for 1 hour with parental strain 14028s, VNP20009 wt, and overexpression mutant strains under the Ptac or PhoPQ promoters, respectively, at MOIs of 10 and 5, followed by gentamicin treatment. The number of invasive bacteria was determined by recording CFU / ml / T0. Results are expressed as the percentage of invasion (CFU / ml / T0). Each infection is represented by the mean of at least two independent experiments + / - SEM. Samples were compared to the VNP20009 control using ordinary one-way ANOVA and Šídák's multiple comparison test.

[0037] Figure 6 Validation of SpiC overexpression strains under in vitro conditions. Relative RNA expression of the spiC gene in VNP20009 wt, ΔspiC, and spiC overexpression mutant strains grown in Mi minimal medium supplemented with 10 μM or 10 mM magnesium sulfate was measured. Fold change was calculated relative to the rpoD housekeeping gene. The gmk gene was used as a control. Each experiment is represented by the mean of two independent experiments + / - SEM. Two-way ANOVA and Dunnett's multiple comparison test were used to compare the samples with the VNP20009 control.

[0038] Figure 7A-D. Preliminary behavioral data in FVB / N mice showed low toxicity levels in the liver and spleen of mice injected with the spiC mutant strain. A. Time-dependent survival of mice injected with different doses of VNP2009, ΔspiC, and PBS. Long-rank (Mantel-Cox) tests were used to compare samples (n=3 mice / group). Intraperitoneal injection of PBS, two different bacterial doses (2x10⁻¹²) 5 (B) 2x10 6 (C) Body weight was monitored over time in the case of VNP20009 wt and ΔspiC mutant strains under CFU. Data are shown as percentage weight loss (n=3 mice / group). Two-way ANOVA with repeated measures and Tukey's test were used to compare mice injected with VNP20009 and ΔspiC to PBS controls. Data are shown as mean + / - SEM. Blue asterisks depict statistical significance comparisons with PBS, and black asterisks depict comparisons with VNP20009 samples. D. Liver (left) and spleen (right) tissues from mice injected with VNP2009 and ΔspiC were weighed on day 11 post-intraperitoneal injection and compared to PBS-injected control mice. Figures show tissue weight in grams (n=3 mice / group). Samples were compared using ordinary one-way ANOVA and Tukey's multiple comparison test. Data are shown as mean + / - SEM. Detailed Implementation

[0039] Bacterial-mediated cancer therapy (BCT) using Gram-negative organisms was introduced by William Coley in the mid-19th century, when he used live Streptococcus pyogenes to treat solid tumors. 17 The basic principle of bacterial cancer therapy is based on certain bacterial strains (including Gram-negative bacteria such as Salmonella typhimurium). 18,19 It can specifically hobby and proliferate in the hypoxic regions of angiogenic tumors, inducing direct lysis of tumor cells and the establishment of anti-tumor immune responses. 20 Furthermore, bacterial injection into tumors has been shown to be anti-angiogenic. 21,22 Therefore, in addition to directly inducing cancer cell death, bacteria can act as immuno-oncologists and anti-angiogenic agents, targeting highly vascularized tumors and establishing immune controls to prevent the development of new tumors.

[0040] A large amount of preclinical and clinical data supports BCT as an immunotherapy strategy. 23,24 Furthermore, for 40 years, intravesical application of a live attenuated strain of Mycobacterium bovis has been the only FDA-approved treatment for orthotopic bladder cancer.25 BCT using attenuated strains of Salmonella typhimurium has shown significant efficacy in several preclinical cancer models. 23,26,27 Early clinical trials of attenuated Salmonella Typhimurium-based BCTs delivered intravenously, directly intratumorally, or orally have shown safety but failed to demonstrate efficacy. 24,28-30 This lack of potency may be due to the rapid division of cancer cells and the use of intravenous delivery. Bacterial inoculants are limited by the toxicity of systemic delivery, and in these trials, the lack of potency may be dose-related. Currently, there is a live attenuated strain of BCT: Mycobacterium bovis tuberculosis, approved by the U.S. Food and Drug Administration, which has been the standard of care for high-risk non-muscle-invasive bladder cancer for the past 40 years. 25 .

[0041] However, BCT has never been recommended as a potential treatment for benign growths, possibly because benign tumors tend to be immunologically cold. 31,32 Therefore, bacterial therapy has never been tested in the context of slow-growing benign tumors such as schwannomas, where traditional cancer therapies, which primarily target highly replicating cells, are ineffective. This article presents a preclinical study supporting bacterial therapy for schwannomas (a benign growth of the peripheral nervous system). It is hypothesized that intratumoral injection of attenuated Salmonella Typhimurium may have the potential to directly kill schwannomas cells, inhibit angiogenesis, and transform the relatively 'cold' to 'hot' immunotumor microenvironment. Further hypothesizing that immune cell death (if it occurs), the generation of a pro-immunogenic tumor environment, and the combination of VEGF / angiogenesis inhibition can synergistically generate an adaptive antitumor immune response.

[0042] To test these hypotheses, in WO2020 / 176764, the effects of two attenuated Salmonella Typhimurium strains (VNP20009 and AppGpp) were evaluated in both xenograft human NF2 schwannoma models in nude mice and allogeneic NF2-schwannoma models in immunocompetent FVB / N mice. Data showed that intratumoral injection of attenuated Salmonella Typhimurium controlled schwannoma growth in both models. Intratumoral injection of Salmonella Typhimurium into schwannomas led to tumor cell killing and induced a systemic antitumor adaptive immune response in immunocompetent mice. This antitumor immune response controlled the growth of non-bacterially injected tumors present during bacterial therapy and prevented the development of "rechallenge" tumors after treatment. Salmonella typhimurium increases tumor-infiltrating CD4+ helper T cells and CD8+ cytotoxic T cells in allogeneic schwannomas that have been injected with the bacteria, contralaterally without injection, and re-challenged (besides its effect on CD4+ cells), and decreases CD25+ cells. Tregs further support the existence of an antitumor adaptive immune response. Intratumoral injection of Salmonella typhimurium increased systemic PD-1 immune checkpoint inhibition, enhancing control of schwannomas in both bacterially injected and contralaterally uninjected tumors, rather than in re-stimulated tumors. Tumor-infiltrating lymphocyte (TIL) studies showed an increase in the number of CD4+ helper T cells and CD8+ cytotoxic T cells, and a decrease in the number of CD25+ regulatory T cells, in schwannomas injected with attenuated Salmonella typhimurium.

[0043] Bacterial-mediated cancer therapy is attractive because it can selectively target and colonize the tumor microenvironment. 51-53 The attenuated strain of Salmonella Typhimurium VNP20009 is one of the most characteristic strains to date for bacterial-mediated cancer therapy and has been evaluated in several solid tumor models. 54,55 The success of VNP20009 in preclinical studies facilitated a Phase 1 clinical trial, in which VNP20009 was administered intravenously to patients with metastatic cancer. This study reported that even with observed tumor colonization, tumor regression was not achieved at the highest tolerated dose. 24Therefore, in this study, we focused on engineering the VNP20009 strain to optimize tumor specificity and biosafety. Our aim was to improve specificity by generating bacteria with enhanced binding and invasion of schwannomas. Therefore, we targeted the SPI-1 T3SS of *Salmonella typhimurium*, which is responsible for invasion of non-phagocytic host cells. We deleted the sipB gene (a component of the SPI-1 needle-like structure) and evaluated its effect on bacterial treatment outcomes for schwannomas. △The sipB *Salmonella typhimurium* only partially eliminated tumor regression in a mouse syngeneic model, indicating that host invasion by *Salmonella typhimurium* is multifactorial and that altering a single component is insufficient to achieve the desired tumor specificity.

[0044] The tumor microenvironment, particularly macrophages, plays a positive role in a mouse model of Salmonella typhimurium used for the treatment of melanoma. 56 In previous studies, we have shown that the invasion and survival of bacteria within macrophages are crucial for their antitumor efficacy. Furthermore, bacteria within phagocytes can spread to other parts of the body, such as the spleen and liver. 38,57,58 Given that SPI-2 T3SS is activated in phagosomes and requires survival within macrophages, we explored the effects of SPI-2 T3SS in the treatment of schwannomas in mice. We deleted the spiC gene from the VNP20009 chromosome and evaluated bacterial load and antitumor potency in two schwannomas mouse models. The antitumor activity of ΔspiC was comparable to that of wild-type VNP20009 in both syngeneic and xenograft mouse models. Interestingly, when assessing bacterial load in syngeneic models, we reported lower colonization in tumor-bearing mice treated with the ΔspiC strain. Therefore, the ΔspiC strain is as effective as VNP20009 in treating mice with schwannomas, while being more attenuated.

[0045] Since in vivo toxicity remains a concern in the clinical translation of VNP20009-mediated immunotherapy, we evaluated the biosafety of the ΔspiC strain in FVB / N mice. We showed that mice systemically injected with ΔspiC exhibited less weight loss, higher survival rates, and less tissue damage compared to mice treated with VNP20009. Three days after injection, ΔspiC showed decreased fitness in spleen and liver tissues, indicating a favorable safety profile observed in mice. Given that higher doses of the bacteria may lead to improved antitumor specificity, we demonstrated that ΔspiC can be safely administered to mice at higher doses than VNP20009, which could improve antitumor specificity and make ΔspiC a better candidate for clinical application.

[0046] Notably, Pawelek et al. have shown that deletion of the SPI-2 gene in VNP20009 eliminates tumor growth inhibition, indicating that the SPI-2 locus is essential for the antitumor effect of Salmonella typhimurium in a mouse melanoma model. 59 They also showed that deletion of several SPI-2 genes did not lead to a significant reduction in virulence. This finding contradicts our ΔspiC attenuation data and general knowledge of SPI-2 function in systemic diffusion in mouse models. 38,60-62 Nevertheless, the authors did not rule out the possibility of compensatory mutations resulting from the accidental amplification of the SPI-2 mutant within tumors. Furthermore, Salmonella typhimurium utilizes different intracellular transport pathways within tumor cells, suggesting that the tumor microenvironment plays a crucial role in the antitumor activity of Salmonella typhimurium.

[0047] The mechanisms by which △spiC strains exert their effects within tumors are not fully elucidated. Uchiya et al. reported that SpiC is involved in flagellar synthesis, indicating that alterations in SpiC levels affect the motility of Salmonella spp. and thus influence virulence in a Salmonellosis model. 63 Although VNP20009 is nonmotile, its flagellated mechanism is fully expressed, making it possible for SpiC to alter host pathway activation by regulating flagellate protein expression, thereby changing the immune response. Furthermore, we previously reported that VNP20009 shifts the macrophage homeostasis from M2 tumorigenesis to M1 tumoricidal activity. 12 Therefore, we hypothesize that ΔspiC may have a more significant effect on macrophage polarization. Further research is needed to investigate the mechanism of the antitumor effect of ΔspiC bacteria in schwannomas.

[0048] Finally, in this study, we developed and evaluated the antitumor potency and safety profile of a novel attenuated Salmonella typhimurium strain in a preclinical mouse model of schwannoma. These data support our efforts to enhance bacterial therapies for schwannoma using bacterial strains that maintain their antitumor potency while possessing an enhanced safety profile.

[0049] Treatment

[0050] As shown in this article, the ΔspiC mutant strain of attenuated Salmonella typhimurium is effective in treating a mouse model of schwannoma.

[0051] Schwannomas are genetically stable, slow-growing, highly vascularized, and have large hypoxic areas. 34These characteristics make schwannomas an ideal homing environment for bacteria and a potentially perfect target for bacterial cytotoxicity and anti-angiogenic properties. Furthermore, the ability of bacteria to induce an immune response allows for the treatment of multiple distant lesions and the establishment of control mechanisms to prevent the recurrence of new schwannomas (a hallmark of these tumors) throughout a patient's life.

[0052] The methods described herein include those for treating benign nervous system tumors and for reducing the risk of having benign nervous system tumors (subjects at risk include those with conditions associated with the development of these tumors, including neurofibromatosis type 1 (NF1); NF2-associated schwannomatosis (NF2 or NF2-SWN); schwannomatosis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannomas; neurofibroma; neurofibromatosis (NF)). In some embodiments, the tumor is a schwannoma. Schwannomas are composed of Schwann cells and form along peripheral nerves, spinal nerves, and cranial nerves. These tumors can cause pain, sensory / motor dysfunction, and death by compressing peripheral nerves, the spinal cord, and / or the brainstem. Multiple schwannomas in peripheral distal nerves and intracranial nerves are hallmarks of neurofibromatosis 1 and 2 (NF1 and NF2) and schwannomatosis (three types of schwannoma). Schwannomas are benign tumors composed of tumor-dedifferentiated Schwann cells. Although usually non-malignant and slow-growing, these tumors can have devastating consequences for patients. They cause extreme pain and impair sensory / motor function, including hearing and vision. Schwannomas in NF2 are often associated with neurological deficits such as sensory abnormalities, weakness, or hearing loss, and similar tumors in schwannomatosis frequently cause intense pain. Some schwannomas become very large, causing compression of adjacent organs or structures and potentially leading to paralysis or death due to progressive spinal cord or brainstem compression. Schwannomas can occur sporadically without exhibiting any genetic characteristics of NF1, NF2, or schwannomatosis. Most vestibular schwannomas are sporadic, hence their high incidence. Vestibular schwannomas typically occur as a single tumor rather than as multiple tumors spreading throughout the body. In some implementations of any aspect, subjects requiring treatment for schwannomas may be subjects who have or have been diagnosed with a condition selected from the following groups: neurofibromatosis type 1 (NF1); NF2-associated schwannomatosis (NF2); schwannomas; meningiomas; schwannomas; schwannomas; vestibular schwannomas; sporadic schwannomas; neurofibrosarcomas; neurofibromas; neurofibromatosis (NF); malignant peripheral nerve sheath tumors; and combinations thereof. Subjects who can be treated using this method include mammals, such as humans and non-human veterinary subjects, such as cats, dogs, horses, goats, and cattle. The current standard of care for patients with NF2 and schwannomas is surgical resection or radiosurgery of symptomatic tumors to reduce tumor size.Unlike sporadic schwannomas (where there is usually only a single tumor, and surgery is generally an effective treatment strategy as long as the lesion is resectable), in schwannomatosis and NF2, where multiple tumors are present, resection often fails due to the difficulty in accessing many tumors and the risk of nerve damage, including major motor dysfunction, significant sensory loss (including deafness in the case of NF2 vestibular schwannomas), and neuropathic pain. Therefore, for most individuals, there is a significant prevalence of schwannomas associated with both NF2 and schwannomatosis, and with current treatment options. This combination of suffering and debilitating effects with a lack of treatment options makes the treatment of schwannomas a major unmet medical need.

[0053] Typically, the method involves administering a therapeutically effective dose of attenuated Salmonella spp. (e.g., Salmonella Typhimurium as described herein) optionally in combination with a checkpoint inhibitor to a subject who requires or has been identified as requiring such treatment. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, and intratumoral (it) administration. In a preferred embodiment, the intratumoral route is used to maximize the bacterial dose and minimize potential dose-limiting toxicities (DLTs). Those skilled in the art will be able to identify the subject as having a benign neurological tumor. In some embodiments, the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the group consisting of: neurofibromatosis type 1 (NF1); neurofibromatosis type 2 (NF2); schwannoma; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof. In some embodiments, the subject does not have a malignant solid tumor, such as cancer. In some implementations, the subject has a condition associated with an increased risk of benign nervous system tumors, such as neurofibromatosis type 1 (NF1); neurofibromatosis type 2 (NF2); or schwannoma.

[0054] As used herein, the term "effective amount" refers to the amount of composition required to alleviate at least one or more symptoms of a disease or condition, and relates to an amount of pharmaceutical composition sufficient to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to an amount of composition sufficient to provide a specific antitumor effect when administered to a typical subject. Effective amounts as used herein will also include, in various cases, an amount sufficient to delay the development of symptoms of a disease, alter the course of symptoms of a disease (e.g., but not limited to slowing the progression of symptoms of a disease), or reverse the symptoms of a disease. Therefore, specifying an exact "effective amount" is generally impractical. However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experiments. Administration of a therapeutically effective amount of the compounds described herein for the treatment of benign nervous system tumors may, for example, result in a reduction in tumor size, tumor number, tumor growth rate, or likelihood of recurrence after treatment with the methods described herein.

[0055] Therefore, this method involves administering attenuated Salmonella Typhimurium strains to inhibit tumor growth. As shown herein, to enhance the efficacy of the therapy, in a preferred embodiment, this method may utilize intratumoral injection of the bacteria instead of intravenous delivery, which increases the bacterial concentration within the tumor and minimizes systemic toxicity. As shown herein, direct injection of attenuated Salmonella Typhimurium into schwannomas has a vaccine-like effect, inducing an antitumor adaptive immune response.

[0056] Attenuated Salmonella Typhimurium

[0057] As used herein, the term “attenuated” refers to a strain that exhibits lower virulence compared to the natural strain, thus becoming harmless or less virulent. Attenuation does not imply inactivation. In both strains, attenuation reduces the likelihood of pathogenicity, including septic shock. While current data primarily concern the spiC deletion mutant of strain VNP20009, other attenuated strains may also be used. Methods for generating attenuated Salmonella typhimurium strains are known in the art and include directed or random mutagenesis followed by screening for reduced virulence. Directed mutations may be used, for example, in the aroA gene (aroA is part of the shikimic acid pathway that links glycolysis to the synthesis of aromatic amino acids; aroA-deficient Salmonella strains are described, for example, in Feiger et al., mBio, 2016, 7:e01220-16); the pul gene (purine synthesis defect); or the asd gene (aspartate-semialdehyde dehydrogenase defect required for cell wall synthesis). Attenuated strains of Salmonella have been disclosed in WO 2014 / 005683; WO 2016 / 202459; WO 2013 / 09189 and US 20200038496 (attenuated S. typhi Ty21a). The strains that can be used in this method include attenuated versions of the following strains: *Salmonella enterica* serovar Typhimurium (“S. Typhimurium”), *Salmonella montevideo*, *Salmonella enterica* serovar Typhi (“S. typhi”), *Salmonella enterica* serovar Paratyphi B (“S. paratyphi B”), *Salmonella enterica* serovar Paratyphi C (“S. paratyphi C”), *Salmonella enterica* serovar Hadar (“S. hadar”), and *Salmonella enterica* serovar Enteritis (“S. typhi”). Salmonella enteriditis (“S. enteriditis”), Salmonella enterica serovar Kentucky (“S. kentucky”), Salmonella enterica serovar Infantis (“S. enteriditis”).Salmonella enterica serovar Pullorurn (“S. pullorum”), Salmonella enterica typhoid serovar Gallinarum (“S. gallinarum”), Salmonella enterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovar Anatum (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S. derby”), and Salmonella enterica cholerae serovar kunzendorf (“S. cholerae”). kunzendorf”) and enteric Salmonella enterica serovar minnesota (“S. minnesota”). See, for example, WO / 2008 / 039408 and US 20200023053; US 20190153452; US20170333490 and US 20180339032; Grant et al., PLoS Pathog. Dec 2012; 8(12): el003070; Tennant and Levine, Vaccine. June 19, 2015; 33(03): C36-C41.

[0058] In a preferred embodiment, the attenuated strain used in this method does not contain Clostridium novie (see, for example, WO2014160950). In a preferred embodiment, the attenuated strain used in this method does not contain a cleavage gene or cassette operatively linked to an intracellularly induced Salmonella promoter (see, for example, US 20170333490).

[0059] combination therapy

[0060] This method may include administration of attenuated Salmonella strains in combination with one or more other treatments. For example, a study disclosed in WO2020 / 176764 showed that intratumoral VNP20009 in schwannomas in immunocompetent mice led to an increase in the percentage of tumor helper CD4+ T cells and cytotoxic CD8+ T cells, and a corresponding decrease in the percentage of CD25+ Tregs. These changes in the tumor-infiltrating T cell population, along with the conversion to M1 tumor-killing macrophages, indicate that Salmonella typhimurium induces an adaptive antitumor immune response. High PD-L1 expression reported in schwannomas indicates resistance to cell-mediated immunity in the tumor immune microenvironment.

[0061] Therefore, in WO2020 / 176764, the efficacy of increased PD-1 immune checkpoint inhibition in controlling the growth of intratumoral Salmonella typhimurium (VNP20009)-associated schwannomas and the role in the development of host anti-tumor adaptive immunity were evaluated. Data showed that this combination led to enhanced tumor regression in bacterially injected schwannomas, which was associated with an increase in the number of CD4+ helper T cells and CD8+ cytotoxic T cells, and a decrease in the number of CD25+ regulatory T cells infiltrating both bacterially injected and uninjected tumors.

[0062] Therefore, this method may include (together or separately) administering a combination of bacteria and a checkpoint inhibitor, such as an inhibitor of PD-1 signaling, for example, an antibody that binds to PD-1, CD40, or PD-L1, or an inhibitor of Tim3 or Lag3, for example, an antibody that binds to Tim3 or Lag3, or an antibody that binds to CTLA-4. Exemplary anti-PD-1 antibodies that may be used in the methods described herein include those that bind to human PD-1; an exemplary PD-1 protein sequence is provided with NCBI accession number NP_005009.2. Exemplary antibodies are described in US8008449; US9073994 and US20110271358, including PF-06801591, AMP-224, BGB-A317, BI 754091, JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, avelumab, pidilizumab, and atezolizumab. Exemplary anti-CD40 antibodies that can be used in the methods described herein include those that bind to human CD40; exemplary CD40 protein precursor sequences are provided with NCBI accession numbers NP_001241.1, NP_690593.1, NP_001309351.1, NP_001309350.1 and NP_001289682.1. Exemplary antibodies include those described in WO2002 / 088186; WO2007 / 124299; WO2011 / 123489; WO2012 / 149356; WO2012 / 111762; WO2014 / 070934; US20130011405; US20070148163; US20040120948; US20030165499 and US8591900, including dacetuzumab, lucarumumab, bleselumab, teneliximab, ADC-1013, CP-870, 893, and Chi Lob. 7 / 4, HCD122, SGN-4, SEA-CD40, BMS-986004, and APX005M. In some embodiments, the anti-CD40 antibody is a CD40 agonist rather than a CD40 antagonist. Exemplary CTLA-4 antibodies that can be used in the methods described herein include those that bind to human CTLA-4; an exemplary CTLA-4 protein sequence is provided with NCBI accession number NP_005205.2.Exemplary antibodies include those described in Tarhini and Iqbal, Onco Targets Ther. 3:15-25 (2010); Storz, MAbs. Jan 2016; 8(1):10-26; US2009025274; US7605238; US6984720; EP1212422; US5811097; US5855887; US6051227; US6682736; EP1141028 and US7741345; and include ipilimumab, tremelimumab and EPR1476. Exemplary anti-PD-L1 antibodies that can be used in the methods described herein include those that bind to human PD-L1; exemplary PD-L1 protein sequences are provided with NCBI accession numbers NP_001254635.1, NP_001300958.1, and NP_054862.1. Exemplary antibodies described in US20170058033; WO2016 / 061142A1; WO2016 / 007235A1; WO2014 / 195852A1, and WO2013 / 079174A1 include BMS-936559 (MDX-1105), FAZ053, KN035, atezolizumab (Tecentriq, MPDL3280A), avelumab (Bavencio), and durvalumab (Imfinzi, MEDI-4736). Exemplary anti-Tim3 (also known as hepatitis A virus cell receptor 2 or HAVCR2) antibodies that can be used in the methods described herein include those that bind to human Tim3; an exemplary Tim3 sequence is provided with NCBI accession number NP_116171.3. Exemplary antibodies are described in WO2016071448; US8552156; and US PGPub. No. 20180298097; 20180251549; 20180230431; 20180072804; 20180016336; 20170313783; 20170114135; 20160257758; 20160257749; 20150086574; and 20130022623, and include LY3321367, DCB-8, MBG453, and TSR-022. Exemplary anti-Lag3 antibodies that can be used in the methods described herein include those that bind to human Lag3; an exemplary Lag3 sequence is provided with NCBI accession number NP_002277.4.Exemplary antibodies are described in Andrews et al., Immunol Rev. 2017 Mar;276(1):80-96; Antoni et al., Am Soc Clin Oncol Educ Book. 2016;35:e450-8; US PGPub. No. 20180326054; 20180251767; 20180230431; 20170334995; 20170290914; 20170101472; 20170022273; 20160303124, and include BMS-986016.

[0063] This method may also include (together or separately) the administration of a combination of bacteria and angiogenesis inhibitors. Many angiogenesis inhibitors are known, including those that target vascular endothelial growth factor (VEGF), its receptor (VEGFR), or other molecules involved in angiogenesis. Specific examples include axitinib (INLYTA); bevacizumab (AVASTIN); cabozantinib (COMETRIQ); everohmus (AFINITOR); lenahdomide (REVLEVHD); lenvatinib mesylate (LENVIMA); pazopanib (VOTRIENT); ramucirumab (CYRAMZA); regorafenib (STIVARGA); sorafenib (NEXAVAR); sunitinib (SUTENT); thalidomide (SYNOVIR, THALOMID); vandetanib (CAPRELSA); or aflibercept (ZALTRAP). See, for example, Zhang et al., ExpNeurol. 2018 Jan;299(Pt B):326-333; de Vries et al., Otol Neurotol. 2015 Aug;36(7):1128-36; Lim et al., Cancer Treat Rev. 2014 Aug;40(7):857-61; Blakeley, CurrOpin Otolaryngol Head Neck Surg. 2012 Oct;20(5):372-9; Goel et al., Cold SpringHarb Perspect Med. 2012 Mar;2(3):a006486.

[0064] Optionally or additionally, this method may be used in conjunction with surgical resection. For example, in some embodiments of any aspect, an attenuated Salmonella strain as described herein may be administered before, simultaneously with, or after surgical resection or partial resection of a growth or tumor (e.g., a schwannoma). Various treatment methods of the present invention may further include treating the subject with surgery, radiation therapy, or chemotherapy, or combinations thereof.

[0065] Pharmaceutical Compositions and Administration

[0066] The methods described herein involve pharmaceutical compositions using attenuated Salmonella spp. as described herein as an active ingredient. Pharmaceutical compositions typically contain pharmaceutically acceptable carriers. As used herein, "pharmaceuticalally acceptable carriers" include saline solutions, solvents, and dispersion media compatible with drug administration. Complementary active compounds may also be incorporated into the composition, such as checkpoint inhibitors and / or angiogenesis inhibitors, as known in the art and / or discussed herein.

[0067] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, and intratumoral administration.

[0068] Example

[0069] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0070] Materials and methods

[0071] The following materials and methods are used in the embodiments described below.

[0072] bacterial strains

[0073] Salmonella Typhimurium VNP20009 (YS1646; ATCC Cat. BAA-3199) and Escherichia coli strains were routinely grown aerobically in LB broth or on LB plates at 37°C. Antibiotics were added to the culture medium at the following concentration: 0.1 mg / mL. -1 Streptomycin and 0.1 mg / ml carbenicillin.

[0074] Production of Salmonella Typhimurium strains

[0075] Using the previously described method 47Deletions of the spiC and sipB genes in *Salmonella typhimurium* were generated. The plasmids used are listed in Table 1 below. In brief, to generate gene deletion vectors, a 15-nucleotide flanking region of approximately 600 bp containing the coding sequence (CDS) of the gene of interest was amplified and cloned into the pRE107 suicide vector using Gibson Assembly (New England Biolabs). To generate spiC overexpressing strains, the phoN neutral site frame was deleted and replaced with spiC CDS under the control of the Tac promoter (pTac) or the phoPQ activating promoter (PphoPQ), as described above. A 15-nucleotide 600 bp flanking region containing the phoN CDS, pTac or PphoPQ, and spiC CDS was amplified by PCR and cloned into the pRE107 suicide vector. The gene of interest was deleted by introducing the corresponding suicide vector into VNP20009 via bacterial conjugation. The plasmid integration into the correct position on the chromosome was verified by PCR.

[0076] Table 1. Plasmids used

[0077]

[0078] Edwards, RA, Keller, LH & Schifferli, DM Improved allelicexchange vectors and their use to analyze 987P fimbria gene expression. Gene207, 149-157, doi:10.1016 / s0378-1119(97)00619-7 (1998).

[0079] Invasion Measurement

[0080] As mentioned above 48Invasion studies were conducted. In short, overnight cultures of *Salmonella typhimurium* were diluted 100:1 in LB broth and aerobically grown at 37°C until OD600 = 0.8 (mid-log phase). Standardized cultures were incubated with monolayers of Caco-2, RAW274.6 macrophages, or HEI-193 schwannomas at multiples of infection of 5:1, 10:1, and 50:1, respectively, at 37°C in 5% CO2 for 1 hour. After co-incubation for 1 hour, cells were washed three times with PBS and incubated for 30 minutes at 37°C in 5% CO2 with medium containing 50 μg / ml gentamicin. Following antibiotic treatment, cells were washed three times with DMEM and lysed with 1% Triton. CFU per ml were calculated by platenting serially diluted cell lysates onto LB broth.

[0081] qPCR

[0082] As mentioned above 49 RNA was extracted from the cultured bacteria. In short, overnight cultures of *Salmonella typhimurium* were diluted 100:1 in LB syrup and incubated at 37°C until mid-logarithmic growth (OD). 600 =0.8-1). Bacteria were collected from 2 mL of culture and resuspended in 1 mL of TRIzol (Thermo Fisher Scientific). RNA was then purified using the PureLink RNA mini kit (Invitrogen) and treated with the Turbo DNA-free kit to remove any contaminating DNA (Ambion). RNA was quantified using Qubit (Thermo-Fisher Scientific) and qRT-PCR was performed using a Kapa SYBR FastOne-Step Universal qRT MasterMix thermal cycler (Eppendorf Mastercycler RealPlex2). For each strain, RNA levels were normalized to rpoD levels.

[0083] Motion performance testing

[0084] Our swarming assay was performed by inoculating 3 µl of standardized overnight bacterial culture into LB medium containing 0.3% (w / v) agar and incubating at 30°C for 12 hours. Spread of bacterial growth a few centimeters from the inoculation point was considered a positive motility assay. The assay was repeated in triplicate.

[0085] Purine auxotroph assay

[0086] For purine auxotroph assays, overnight cultures of *Salmonella typhimurium* were diluted and aerobically grown at 37°C in M9 minimal medium or M9 medium supplemented with 20 µg / ml adenine. Optical density (OD) at 600 nm was measured using a Synergy plate reader. 600 ).

[0087] animal

[0088] All animal experiments were approved and conducted under the supervision of the Institutional Animal Care and Use Committee of Massachusetts General Hospital (MGH, Boston, MA). Animals (nu / nu and FVB / N mice) were kept in a 12:12 light-dark cycle and had free access to food and water.

[0089] animal models

[0090] We used a glass micropipette and a gas-driven microsyringe (IM-300; Narishige, Tokyo, Japan) to achieve this. 50 As described above 44 3x10 4 Individual HEI-193FC or 1x10 4 Schwannomas were induced by direct injection of 08031-8FC mouse schwannomas into the left sciatic nerve of isoflurane-anesthetized athymic nude mice (nu / nu, 5-7 week old males; National Cancer Institute [NCI]) or syngeneic FVB / N mice (5-7 week old males; Charles River Laboratory). Prior to injection, the cells were trypsin-digested and washed with cold PBS, and each inoculum was suspended in 0.5 µl of PBS. Two weeks after implantation of HEI-193FC cells or one week after implantation of 08031-9FC cells, 10 cells were injected with 10 cells of 10 ... 4 CFU (Chronic Fusarium typhimurium) injection of Salmonella typhimurium into sciatic nerve tumors. We monitored tumor growth of the HEI-193FC tumor weekly using in vivo bioluminescence imaging, and tumor growth of the 08031-9FC tumor twice weekly. 50 We captured images using high-performance IVIS spectroscopy (Caliper Life Sciences, Hopkinton, MA) 10 minutes after intraperitoneal injection of the Fluc substrate d-fluorescein into mice. For the immunocompetent syngeneic subcutaneous (Sc) model, we resuspended 08031-9 cells in PBS and mixed them with Matrigel (1:1) (BD Biosciences), then... 5Tumor cells were subcutaneously transplanted into immunocompetent mice. Tumor volume was estimated using W x L x L x π / 6, where width (W) and length (L) correspond to the two maximum dimensions. 36 Once the tumor reaches 150mm 3 We used an insulin injector to inject either a strain of *Salmonella typhimurium* or a PBS solvent control into the tumor. The excised tumor was homogenized in PBS, and the extract was serially diluted and plated onto LuriaBroth (LB) plates. After incubating the plates overnight at 37°C, *Salmonella typhimurium* colonies were counted, and the bacterial load (CFU / g) was determined as the number of viable bacterial colonies cultured from the homogenized tumor tissue, normalized to the weight of the recovered tissue. 23 .

[0091] Biosafety animal research

[0092] VNP20009 and ΔspiC strains were administered to naïve FVB / N mice via intraperitoneal injection at various doses. Animals were monitored and weighed daily. On day 3 or 11 post-injection, organs were removed, aseptically weighed, and homogenized in 5 ml of sterile PBS. The homogenate was then plated on modified LB medium and incubated at 37°C for 24 hours. Colony-forming units (cfu) per gram of tissue were determined by counting colonies and normalizing them to the weight of the recovered organ.

[0093] Statistical analysis

[0094] All group values ​​are expressed as mean ± standard error of the mean. Details of the statistical tests performed are shown in the corresponding legends. One-way ANOVA (between-subjects factorial) or two-way ANOVA (between-subjects factorial) was used, followed by post-hoc multiple comparison tests. Statistical analysis was performed using Prism software (GraphPad).

[0095] Example 1. Optimization of bacterial binding and invasion of schwannoma cells

[0096] Despite reliable preclinical data supporting VNP20009 as an antitumor agent in various tumor models, clinical trials of VNP20009 for metastatic malignant melanoma have been disappointing. 24,35 In this study, we aimed to enhance tumor specificity by modifying endogenous loci on the Salmonella spp. chromosome to improve the binding and invasion of schwannomas. To support this hypothesis, our previous research has shown that altering Salmonella attachment to tumor cells can enhance tumor specificity. 12Therefore, we targeted Salmonella Typhimurium T3SS, the needle-like structure that directly secretes virulence proteins into host cells. We generated in-frame deletion mutants of the sipB gene (ΔsipB) encoded by the Salmonella spp. pathogenic island (SPI) locus to eliminate the function of SPI-1 T3SS required for epithelial cell invasion and inflammation. When in rich laboratory media such as Lysogeny broth (LB); Figure 4A When cultured in (-B), we did not observe any changes in the growth of strain (△sipB).

[0097] Next, we evaluated the antitumor activity of the ΔsipB strain in a syngeneic mouse schwannoma model. 12,36 We subcutaneously (sc) implanted mouse NF-2 schwannoma tumor cells into syngeneic FVB / N mice, followed by intratumoral injection with PBS, VNP20009, or ΔsipB strain. Tumor growth was assessed over time using direct measurement with calipers. Our data showed that, compared with the PBS control, the ΔsipB strain significantly controlled tumor growth. Figure 1A The tumor growth profiles of mice injected with ΔsipB and PBS differed 2 weeks after intratumoral bacterial injection. Although tumor suppression was enhanced in mice treated with ΔsipB compared to PBS, the greatest tumor control was observed in mice treated with VNP20009. The effect of VNP20009 on tumor volume was significant 10 days after intratumoral bacterial injection, and at the end of the study, VNP20009 reduced tumor growth by approximately two-fold compared to the PBS control. Figure 1A ).

[0098] We then evaluated the ability of Salmonella Typhimurium mutant strains to bind to and invade relevant mammalian cell lines by performing a gentamicin protection assay. After applying parental Salmonella Typhimurium 14028, VNP20009, and ΔsipB to monolayers of Caco-2 epithelial cells, RAW274.3 macrophages, or HEI-193 schwannoma tumor cells, extracellular bacteria were removed with gentamicin. Intracellular bacteria and bacteria attached to host cells were recovered 1 hour later to measure the percentage of bacterial invasion. The data showed that, in any cell line, the absence of sipB did not alter the level of cell invasion compared to VNP20009. Figure 4BTo further investigate this finding, we overexpressed the sipB protein by replacing the phoN neutral site on the Salmonella chromosome with a construct consisting of a Ptac inducible promoter or a phoPQ activating promoter (which is known to be active in vivo) and the sipB gene. We then assessed whether the altered proteomic profile was a cause of improved binding and invasion. As determined by gentamicin assays, overexpression of the SPI-1-encoded sipB protein did not improve binding and / or invasion in any of the test cell lines. Our data suggest that sipB deletion only partially eliminated tumor potency in a syngeneic mouse NF2 schwannoma model, indicating that alterations to the SPI-1 T3SS enhance tumor specificity. Figure 5 Furthermore, given the complexity of the tumor microenvironment, it is not surprising that our in vitro invasion data were not correlated with our in vivo tumor control findings. Therefore, it also suggests that in vitro screening for Salmonella strains targeting tumor cell invasion cannot be used as a predictor of in vivo schwannoma growth control.

[0099] Example 2. Tumor suppression effect of VNP20009 spiC deletion mutant in syngeneic mouse NF2 and xenograft human NF2 mouse models.

[0100] Our primary objective is to develop an attenuated Salmonella typhimurium strain with similar or better antitumor efficacy to VNP20009, but with a superior toxicity profile. SpiC (one of the effectors encoded by the SPI-2 locus and a component of T3SS) is required for the secretion of T3SS secretory proteins. 37 Its absence led to a significant reduction in intestinal colonization in a mouse typhoid fever model. 38 Therefore, we deleted spiC from the VNP20009 chromosome (the mutant is called VNP20009-ΔspiC) and verified that such deletion does not alter bacterial fitness in LB standard medium. Similar to the sipB mutant strain, our data show that the deletion of spiC does not increase the invasion of related mammalian cell lines such as Caco-2, RAW274.3, and HEI-193 cell lines. Figure 4B This finding is consistent with previous observations, in which ΔspiC does not affect invasion but enhances survival within macrophages. 38,39 .

[0101] Next, we evaluated the tumor control and colonization profile of the VNP20009-spiC mutant in syngeneic and xenograft mouse models. First, we induced subcutaneous schwannoma tumors in syngeneic, immunocompetent (FVB / N) mice (08031-9 cell line). Once the tumor size reached approximately 150 mm... 3 We tested the therapeutic effects of intratumoral injection of PBS, VNP20009 wt, or the ΔspiC mutant strain. As expected, intratumoral injection of VNP20009 controlled tumor growth in approximately 10 days. Figure 1B Notably, animals treated with intratumoral injection of the ΔspiC mutant strain showed significantly slower tumor growth compared to mice treated with PBS, and similar antitumor efficacy to mice treated with the attenuated VNP20009 wt strain. Figure 1B ).

[0102] Subsequently, we sought to determine whether intratumoral treatment with *Salmonella typhimurium* overexpressing SpiC (ΔphoN::spiC under the PphoPQ promoter) would reverse the antitumor activity of the ΔspiC mutant strain. First, we validated the SpiC expression level of the overexpressing strain in vitro. It has been demonstrated that the PhopQ activation promoter positively regulates target gene expression in mice. 40 Its function under laboratory conditions was simulated by using a low pH and low magnesium content in the culture medium. 41,42 By analyzing the relative levels of SpiC RNA using qPCR, we confirmed that the SpiC overexpressing strain cultured in a minimal medium containing low magnesium expressed more SpiC transcripts than the VNP20009 control strain cultured under the same conditions. Figure 6 Next, we evaluated the antitumor efficacy of SpiC overexpression of Salmonella typhimurium in a subcutaneous syngeneic schwannoma mouse model. In contrast to animals receiving intratumoral injection of VNP20009 or ΔspiC, treatment with SpiC overexpression of Salmonella typhimurium eliminated tumor suppression, indicating that fine-tuning of SpiC protein levels under test conditions is necessary for clinical efficacy. Figure 1B We also evaluated the bacterial burden profile on days 2, 7, and 12 after intratumoral injection by calculating the tumor colony-forming units (CFU) / g. The bacterial burden of the ΔspiC mutant strain was significantly lower on days 7 and 12 compared to VNP20009 and SpiC overexpressing strains, indicating that a lower dose of the ΔspiC mutant strain is required to achieve a similar potency level as with high-dose treatment with the VNP20009 strain. Figure 1C ).

[0103] To assess the broader applicability of our method, we also examined the antitumor efficacy of the resulting strains in syngeneic and xenogeneic sciatic schwannoma mouse models. We implanted firefly luciferase (Fluc)-expressing tumors from HEI-193FC (human NF2 schwannoma line) and 08031-9FC (mouse NF2-deficient schwannoma line) into the sciatic nerve of thymic null (nu / nu) immunized compromised mice and syngeneic immunized intact (FVB / N) mice, respectively. 43-45 Once the tumor size stabilizes, we use approximately 10 4 Attenuated VNP20009 or ΔspiC mutant bacteria or PBS control were injected into the tumor-bearing sciatic nerve. Tumor growth in the syngeneic model was assessed every 2 days by in vivo bioluminescence imaging, and tumor growth in the xenograft model was assessed weekly. In both tumor models, the tumor growth profiles of the VNP20009 or ΔspiC and PBS groups differed as early as day 7 after intratumoral treatment. Figure 1D and 1F Furthermore, compared with PBS-treated mice, mice treated with VNP20009 and ΔspiC showed significant tumor inhibition. Figure 1D and 1F Clearly, in xenograft models, the VNP20009 and ΔspiC groups resulted in undetectable tumor levels in mice treated in 3 / 5 and 4 / 5, respectively. Figure 1F We also examined the bacterial load within tumors in syngeneic mice. At 22 days post-implantation, we observed approximately 2 x 10⁻⁶ bacteria in the tumor tissue. 8 CFU / g VNP20009 wt, while we detected 3 x 10 4 The CFU / g ΔspiC mutant strain confirmed our previous findings in the subcutaneous syngeneic model. Figure 1E These data indicate that even though the ΔspiC mutant strain colonizes the tumor environment less efficiently, it remains effective in treating schwannomas.

[0104] Example 3. Toxicity and behavior of attenuated ΔspiC strain in FBV / N mice

[0105] Because in vivo toxicity remains a concern in the clinical translation of VNP20009-mediated cancer therapies, we conducted biosafety studies on VNP20009 and the ΔspiC strain. In our pilot experiments, we used PBS, 2x10 5 (low dose), 2x10 6 (Medium dose) or 2x10 7(High-dose) CFU of VNP20009 or ΔspiC strains was administered intraperitoneally to mice receiving FVB / N (N=3). Disease and weight changes were monitored daily. Animals were sacrificed after 11 days to assess organ inflammation by measuring spleen and liver weight. Survival data of mice injected with low or medium doses of VNP20009 or ΔspiC strains were indistinguishable from those of mice injected with PBS controls (100%). Figure 7A However, the highest dose of the VNP20009 strain induced a significant drop-in survival rate, with only 33% of mice surviving 11 days after bacterial injection. Mice injected with the same dose of the ΔspiC strain survived until the end of the study. We calculated the LD50 of the VNP20009 strain in FVB / N mice to be 2 x 10⁻⁶. 7 ( Figure 7A We observed that injections were made with PBS or 2x10 5 Mice with CFU containing VNP20009 or ΔspiC strain showed no weight difference. Figure 7B It is worth noting that 2x10 6 Following CFU injection, the ΔspiC strain caused a temporary decrease in body weight; however, the mice regained their body weight within 24 hours. Injection of 2x10... 6 Mice infected with CFU containing the VNP20009 strain experienced a 10% decrease in body weight at the end of the observation period. Figure 7C ).

[0106] To determine whether the ΔspiC strain induced less organ inflammation in injected mice, we collected livers and spleens from all injected animals at the end of the study and compared tissue weights with those from PBS-injected controls. Tissues extracted from mice injected with either dose of the ΔspiC strain were indistinguishable from those collected from PBS-injected mice. In contrast, mice injected with low and medium doses of VNP20009 showed significantly developed tissue inflammation, as indicated by increased liver and spleen weights. Figure 7D This indicates that genetic ablation of spiC induces less systemic toxicity than wt VNP20009 strain, and is therefore more attenuated in FVB / N mice.

[0107] Next, we investigated the bacterial load at various sites in infected FVB / N mice. Our pilot experiment with mice injected with the highest dose of VNP20009 showed that 66% of the animals died within 4 days of injection; therefore, we chose day 3 post-injection as the early time point. We administered PBS intraperitoneally or 2x10 7CFU of VNP20009 or ΔspiC strains. We monitored weight loss and showed an initial decrease in body weight over 24 hours in both groups of mice injected with the Salmonella typhimurium strain. However, by day 3, mice injected with ΔspiC gained more body weight than mice injected with the same dose of VNP20009. Figure 2A Liver and spleen samples collected from mice injected with VNP20009 were highly colonized, with an average of 10% in each liver and spleen. 6 and 10 7 CFU / g. In both tissues, the ΔspiC loading was significantly lower than that of VNP20009, indicating that SpiC does indeed affect colonization in tumors and systemic sites. Figure 2B Liver and spleen weight measurements on day 3 post-injection showed no significant difference between VNP20009 and ΔspiC strains, suggesting that systemic inflammation may occur at a later point. Figure 2C -D).

[0108] To confirm our findings, we repeated the biosafety study with more animals in each group and assessed the bacterial load on day 10 post-injection (Figure 3). PBS or two different doses (2x10⁻¹²) were used. 6 and 2x10 7 Intraperitoneal injection of VNP20009 or ΔspiC Salmonella strains (CFU) into FVB / N mice, with disease monitored over time. As previously observed, at 2 x 10 6 Or 2x10 7 Intraperitoneal administration of CFU doses of ΔspiC strain to mice did not produce mortality. Figure 3A Consistent with our previous research, 2x10 7 CFU's VNP20009 was sufficient to kill 66% of injected mice, confirming the previously established LD50 ( Figure 3A ) 2x10⁻⁶ was observed within 48 hours after injection. 6 Mice with CFU ΔspiC experienced a decrease in body weight, however, the animals regained body weight at the end of the observation period. Figure 3B On day 10 post-injection, liver and spleen were collected, and bacterial load was calculated as CFU per gram of tissue. VNP20009 levels in the spleen and liver reached approximately 2 x 10⁻⁶. 3 and 2x10 4 CFU indicated that tissue colonization of both strains on day 10 was lower than that observed on day 3 post-infection, and no significant difference was observed between VNP20009 and ΔspiC. Figure 3CFinally, we assessed tissue inflammation by measuring tissue weight and comparing it with healthy control tissue. As previously observed, tissue extracted from mice injected with VNP20009 was heavier than tissue from mice injected with ΔspiC, confirming that ΔspiC is less toxic than wild-type VNP20009. Figure 3D In summary, our data suggest that the ΔspiC strain colonizes less extensively throughout the body, making it safer for treating schwannomas in mice.

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[0173] Other embodiments

[0174] It should be understood that although the invention has been described in conjunction with a detailed description, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. A method of treating a subject with or at risk of developing a nervous system tumor, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a live attenuated spiC-deficient strain of Salmonella bacteria, optionally in combination with an immune checkpoint inhibitor and / or angiogenesis inhibitor.

2. The method of claim 1, wherein the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the group consisting of: neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannoma; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof.

3. The method of claim 1, wherein the subject does not have a malignant solid tumor.

4. The method of claim 1, wherein the subject has a condition associated with an increased risk of benign nervous system tumors.

5. The method of claim 4, wherein the condition associated with the increased risk of benign nervous system tumors is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannoma.

6. The method according to claim 1, wherein the attenuated Salmonella is administered intratumorally or intravenously.

7. The method according to claims 1 to 6, wherein the attenuated spiC-deleted variant of Salmonella is an attenuated strain of Salmonella typhimurium.

8. The method according to claim 7, wherein the attenuated spiC-deficient variant of Salmonella typhimurium is enteric Salmonella typhimurium serotype VNP20009.

9. The method of claim 1, wherein the composition does not contain Clostridium novie.

10. The method of claim 1, wherein the attenuated Salmonella does not contain a cleavage gene or cassette operatively linked to an intracellularly induced Salmonella promoter.

11. The method according to claims 1 to 10, wherein the attenuated Salmonella strain is not VNP20009.

12. The method of claim 1, wherein the checkpoint inhibitor is an inhibitor of PD-1 signaling or CTLA-4 signaling.

13. The method of claim 11, wherein the inhibitor of PD-1 signaling is an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

14. The method of claim 1, wherein the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR).

15. The method of claim 11, wherein the VEGF inhibitor is bevacizumab.

16. A composition comprising a live attenuated spiC-deleted variant of Salmonella enterica typhus serotype VNP20009.

17. The use of a composition comprising a live attenuated spiC-deficient variant of Salmonella bacteria, optionally in combination with a checkpoint inhibitor and / or angiogenesis inhibitor, for the treatment of a subject with a benign nervous system tumor or at risk of having a benign nervous system tumor.

18. The composition for the use of claim 17, wherein the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the group consisting of: neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannoma; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof.

19. The composition for the said use according to claim 17, wherein the subject does not have a malignant solid tumor.

20. The composition for the use of claim 17, wherein the subject suffers from a condition associated with an increased risk of benign nervous system tumors.

21. The composition for the use according to claim 20, wherein the condition associated with the increased risk of benign nervous system tumors is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannoma.

22. The composition for the said use according to claim 17, wherein the attenuated Salmonella spp. is formulated for intratumoral or intravenous administration.

23. The composition for the said use according to claims 17 to 22, wherein the attenuated Salmonella is an attenuated strain of Salmonella typhimurium.

24. The composition for the said use according to claim 21, wherein the attenuated strain of Salmonella typhimurium is enteric Salmonella typhimurium serotype VNP20009.

25. The composition for the said use according to claim 17, wherein the composition does not contain Clostridium novie.

26. The composition for the said use according to claim 17, wherein the attenuated Salmonella does not contain a cleavage gene or cassette operatively linked to an intracellularly induced Salmonella promoter.

27. The composition for the said use according to claim 17, wherein the checkpoint inhibitor is an inhibitor of PD-1 signaling or CTLA-4 signaling.

28. The composition for the said use according to claim 27, wherein the inhibitor of PD-1 signaling or CTLA-4 signaling is an antibody that binds to PD-1, CD40, PD-L1 or CTLA-4.

29. The composition for the said use according to claim 17, wherein the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR).

30. The composition for the said use according to claim 17, wherein the VEGF inhibitor is bevacizumab.

Images