EP4683662A2 - Treatment of nervous system tumors using attenuated salmonella typhimurium - Google Patents

Abstract

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

Description

TREATMENT OF NERVOUS SYSTEM TUMORS USING ATTENUATED SALMONELLA TYPHIMURIUM

CLAIM OF PRIORITY

This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/453,202, filed on March 20, 2023. The entire contents of the foregoing are hereby incorporated by reference.

TECHNICAL FIELD

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

BACKGROUND

Schwannomas are slow-growing benign neoplasms derived from Schwann-lineage cells^1,2. Depending on location and size, these tumors can cause a variety of gain- and loss-of- function neurological deficits, including hearing loss, imbalance, tinnitus, motor loss, and severe pain^3,4; in some cases, they can lead to death due to brain stem compression⁵. Schwannomas may arise sporadically (thus, termed “sporadic schwannoma”) or as part of the debilitating genetic syndromes NF2-related schwannomatosis (NF2 or NF2-SWN) and schwannomatosis (SWN)⁶. Treatment of schwannoma is largely limited to operative resection and symptomatic management of pain. Resection, which for many patients is noncurative, is often associated with additional neurologic damage, and may be impractical due to location or large numbers of tumors⁷. Anti-cancer therapeutics have not demonstrated efficacy for schwannomas due to the slow replicating nature of these benign lesions⁸'¹⁰. Bevacizumab is currently the only generally accepted pharmacotherapy for schwannoma; it temporally stabilizes tumor growth by targeting the highly vascularized nature of a subset of these neoplasms^{8 11}. Current strategies for pain control are, unfortunately, inadequate for many thus further increasing the burden of disease. Treatment is further complicated by the fact that schwannomas appear in multiple locations with new lesions developing throughout life. Thus, schwannomas and their associated diseases cause lifelong suffering that cannot be stably controlled with current treatment options. In sum, schwannomas are slow-growing, benign neoplasms that develop throughout the body associated with peripheral nerves, including along the spinal cord and within the cranium. Schwannomas frequently first appear in childhood or adolescence, with new tumors developing throughout life. These tumors cause pain, sensory/motor dysfunction, and death through compression of peripheral nerves, the spinal cord, and/or the brain. The great suffering and debility associated with schwannomas, in conjunction with the paucity of therapeutic options, makes their treatment a major unmet medical need.

SUMMARY OF THE INVENTION

Described herein is a therapeutic approach for benign neoplasms, including schwannoma that involves intratumoral (i t.) injection of one of several attenuated, deletion mutants of a Salmonella Typhimurium (S. Typhimurium).

The term 'deletion mutant' hereafter refers to a strain that was genetically engineered to lack a specific chromosomal DNA sequence required for encoding a specific protein of known function. The S. typhimurium deletion mutants described include strains deleted in a gene called spiC. SpiC encodes a critical component of the Type 3 secretion system (T3SS) of S. typhimurium. The T3SS of S. Typhimurium is a nanomachine used to allow this bacterium to enter into the cytosol of host mammalian cells and then kill the host cell or inhibit its function. These cells include macrophages and other immune cells important to the elimination of tumors. The spiC mutant called the spiC F6-Y129 deletion mutant (or VNP- SspiC or simply \spiC in included figures) is the focus of this invention. The \spiC strain w as found to be highly attenuated in virulence compared to its parental strain VNP20009 (also denoted YS1646) and was also found to be equal in its efficacy as its parental strain for schwannoma tumors in a mouse model for NF2 disease.

The data support therapy via intra-tumoral (i.t.) injection of a live attenuated, spiC F6- Y129 deletion mutant of S. Ty phimurium, optionally in combination with PD-1 checkpoint inhibition, as an immunotherapy capable of controlling growth of bacterially-injected and non-injected benign nervous system tumors including schwannomas and neurofibromatosis- related neoplasms including neurofibromatosis type 1 (NFl)-associated tumors and meningiomas. The data further suggest the potential of the therapeutic strategy⁷ to control growth of tumors that arise following initial treatment. Importantly, direct injection of VNP20009 (the <S’. typhimurium parental strain of the spiC F6-Y129 deletion mutants) into tumors had a vaccine-like action inducing an systemic anti-tumor immune response¹². The spiC F6-Y129 deletion mutant of VNP20009 showed similar efficacy to VNP20009 while also displaying reduced virulence in an acute lethality model for murine systemic disease due to S. typhimurium (FIG. 1)

Thus, provided herein are methods for treating a subject having or at risk of having a benign nervous system tumor (subjects who are at risk include those who have a disorder associated with development of these tumors, including neurofibromatosis type 1 (NF1); NF2-related schwannomatosis (NF2 or NF2-SWN); schwannomatosis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF)). The methods include administering to the subject a therapeutically effective amount of a composition comprising live attenuated, spiC F6-Y129 deletion mutant of <S’. Typhimurium, optionally in combination with an immune checkpoint inhibitor and/or angiogenesis inhibitor. Also provided herein are compositions comprising spiC F6-Y129 deletion mutants of S. Typhimurium live attenuated strains, optionally in combination with a checkpoint inhibitor and/or angiogenesis inhibitor, for use in a method of treating a subject having or at risk of having a benign nervous system tumor.

In some embodiments, the subject is a subject having or diagnosed as having a benign tumor or tumor-associated condition selected from the group consisting of: neurofibromatosis type 1 (NF1); NF2-related schwannomatosis (NF2 or NF2-SWN); schwannomatosis; 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 (i.e., has not been diagnosed with a malignant solid tumor). In some embodiments, the subject has a condition associated with an increased risk of a benign nervous system tumor, e.g., neurofibromatosis type 1 (NF1); NF2-related schwannomatosis (NF2); or schwannomatosis.

In some embodiments, the attenuated, spiC F6-Y129 deletion mutants of S. Typhimurium VNP20009 are administered intratumorally or intravenously. The key embodiment of the spiC F6-Y129 mutation is the fact that it is an internal, in-frame deletion of the spiC gene. This deletion is defined by loss of the codons of the spiC gene that encode from Phenylalanine 6 through Tyrosine 129 of the spiC gene. Thus, the spiC F6-Y129 deletion mutation removes the coding sequence for the SpiC protein between Phenylalanine 6 and Tyrosine 129 of the SpiC protein. This spiC F6-Y129 deletion construct will hereafter be referred to as the spiC F6-Y129 deletion or simply \spiC. Mutant derivatives of all parental strains carrying this genetic alteration in their genomes will be referred to as spiC F6-Y129 deletion mutants or simply \spiC. In some embodiments, the attenuated, spiC F6-Y129 deletion mutants include a derivative of Salmonella enterica serovar Typhimurium strain VNP20009¹³’¹⁶. The genome sequence of VNP20009 defined mutations in genes that alter lipid A acylation (for example, in the msbB gene) or block purine biosynthesis (for example, the purl gene)¹³. A derivative of VNP20009 that carries the spiC F6-Y129 deletion is also called herein VNP-As/wC or simply \spiC in the figures attached to this application.

In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling, e.g., an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

In some embodiments, the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR), e.g., Bevacizumab.

Further, provided herein are methods to treat benign nerve sheath tumors in a mammal comprised of administering to said mammal a therapeutically effective dose or titer of an attenuated strain of pathogenic enteric bacteria. In some embodiments, the attenuated strains include spiC F6-Y129 deletion mutant of S. Typhimurium VNP20009.

Provided herein are methods of a treating a subject having or at risk of having a nervous system tumor, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a live attenuated spiC deletion variant strain of a Salmonella bacteria, optionally in combination with an immune checkpoint inhibitor and/or angiogenesis inhibitor.

Further, provided herein are compositions comprising a live attenuated spiC deletion variant strain of Salmonella bacteria, optionally in combination with a checkpoint inhibitor and/or angiogenesis inhibitor, for use in a method of a treating a subject having or at risk of having a benign nervous system tumor.

In some embodiments, the subject is a subject having or diagnosed as having a benign tumor or tumor-associated condition selected from the group consisting of neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannomatosis; 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. In some embodiments, the subject has a condition associated with an increased risk of a benign nervous system tumor. In some embodiments, the condition associated with an increased risk of a benign nervous system tumor is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannomatosis.

In some embodiments, the attenuated Salmonella is administered intratumorally or intravenously. In some embodiments, the attenuated, spiC deletion variant Salmonella is an attenuated strain of S. typhimurium.

In some embodiments, the attenuated, spiC deletion variant strain of S. typhimurium is Salmonella enterica serovar typhimurium strain VNP20009.

In some embodiments, the composition does not comprise Clostridium novyi.

In some embodiments, the attenuated Salmonella do not comprise a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter.

In some embodiments, the attenuated Salmonella strain is not VNP20009.

In some embodiments, the checkpoint inhibitor is an inhibitor of PD- 1 or CTLA-4 signaling.

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

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

Also provided herein are compositions comprising a live attenuated spiC deletion variant strain of Salmonella enterica serovar Typhimurium strain VNP20009, e g., as described herein.

In some embodiments, the administration includes, but is not limited, intravenous injection or by way of direct injection into the benign nerve sheath tumor. In some embodiments, the nerve sheath tumor includes, but is not limited to, a neurofibroma or schw annoma. In some embodiments, the tumor includes, but is not limited to those associated with Neurofibromatosis type 1, NF2-related schwannomatosis, Schwannomatosis, or sporadic schwannoma. In some embodiments, the methods include administering to said mammal therapeutically effective doses of an attenuated, splice variant strain of pathogenic enteric bacteria and a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor, includes, but is not limited to, a peptide, antibody, small molecule, microRNA, antisense oligonucleotide, or small interfering RNA. In some embodiments, the checkpoint inhibitor is a monoclonal antibody that binds to the epitope of an antigen. In some embodiments, the monoclonal antibody-binding epitope is in a PD-1 or CTFA-4 antigen. In some embodiments, the mammal is a human.

Also provided herein are pharmaceutical compositions comprised of an attenuated, VNP-AstyC 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 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 belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting.

All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF THE FIGURES

FIGs. 1A-F. Evaluation of tumor control by VNP20009 SPI-1 and SPI-2 mutant strains in multiple tumor mouse models, i.t. injection of SPI-1 \sipB (A), \spiC (B) and VNP20009 mutants in NF-2 syngeneic mouse model. Volumes of 08031 implanted tumors were monitored over time by treatment in FVB/N mice and compared to PBS treated mice (n=8/group). Repeated-measured two-way ANOVA with Tukey's multiple comparisons test w as used to compare samples. Data are shown as mean +/- SEM. C. Biodistribution of VNP20009, \spi(' and .s/vC-overexpressing strains at day 2, 7 and 12 after intratumoral injection of bacteria. Bacterial colonization was determined by plating excised and sonicated tissue tumors from the mouse- NF2 syngeneic experiment in LB plates and calculated as CFU/g (n=3/group). One-way ANOVA with Dunnett’s multiple comparisons test was used to compare samples at different time point. Data are shown as mean +/- SEM. I.t. injection of ^spiC and VNP20009 strain mutants in intrasciatic syngeneic (D) and xenograft (F) schwannoma models. Volumes of 08031 (D) and HEI-193 (F) implanted-tumors were monitored over time by treatment in FVB/N (D) and nude (F) mice and compared to PBS treated mice (n=8/group). Repeated-measured two-way ANOVA with Tukey’s test was used to compare tumor signals. Data are shown as mean +/- SEM. E. Biodistribution of VNP20009 and \spi( ' strains at day 22 after intratumoral injection of bacteria. Bacterial colonization was determined by plating excised and sonicated tissue tumors from the intrasciatic syngeneic mouse schwannoma experiment in LB plates and calculated as CFU/g (n=5/group). Mann Whitney test was used to compare samples. Data are shown as mean +/- SEM.

FIGs 2A-D. \spiC mutant shows less toxicity effect in FVB/N mice in comparison to VNP20009 wt strain. A. Body weight was monitored upon i.p. injection of PBS, 2x10⁷ CFU of either VNP20009 wt or spiC mutant strains over 3 days. Data are shown as bodyweight percentage loss (n= 5 mice/group). Repeated-measured two-way ANOVA with Tukey’s multiple comparisons test was used to compare samples. Data are shown as mean +/- SEM. B. Biodistribution of VNP20009 and spiC strains in livers and spleens at day 3 post intraperitoneal injection of bacteria. Bacterial colonization was determined by plating excised and sonicated liver and spleen tissues in LB plates and calculated as CFU/g (n=5/group). Paired t-test was used to compare samples. Data are shown as mean +/- SEM. C-D. Liver and spleen tissues from VNP2009 and A^zC-injected mice, were weighted at day 3 post i.p. injection and compared to PBS-injected control mice. Graphs are showed was grams of tissue (n= 5 mice/group). Ordinary one-way ANOVA with Tukey’s multiple comparisons test was used to compare samples. Data are shown as mean +/- SEM.

FIGs. 3A-D. \spiC mutant shows less toxicity effect in FVB/N mice compared to VNP20009 wt strain. A. Survival rate of VNP2009, spiC and PBS-injected mice over rime. Long-rank (Mantel-Cox) test w as used to compare samples (n= 5 mice/group). B. Body weight was monitored upon i.p. injection of PBS, 2xl0⁶ CFU VNP20009 wt and \spiC mutant strains over 10 days. Data are shown as body weight percentage loss (n= 5 mice/group). Repeated-measured two-way ANOVA with Tukey’s multiple comparisons test was used to compare VNP20009 and \spi(' injected mice to PBS controls. Data are shown as mean +/- SEM. C. Biodistribution of VNP20009 and \ piC strains in livers and spleens at day 10 post intraperitoneal injection of 2x10⁶ CFU bacteria. Bacterial colonization was determined by plating excised and sonicated liver and spleen tissues in LB plates and calculated as CFU/g (n=5/group). One-way ANOVA with Tukey’s multiple comparisons test was used to compare samples. Data are shown as mean +/- SEM. D. Liver (left panel) and spleen (right panel) tissues from VNP2009 and spiC-injected mice were weighed at day 3 post i.p. injection and compared to PBS-injected control mice. Graphs are showed was grams of tissue (n= 5 mice/group). Ordinary one-way ANOVA with Tukey’s multiple comparisons test w as used to compare samples. Data are shown as mean +/- SEM.

FIGs. 4A-B. Deletion of SPI-1 and SPI-2 S. Typhimurium VNP20009 genes does not impair growth in standard lab media and invasion in relevant cell lines. A. Growth of VNP20009 and mutant derivates was monitored over time by measuring optical density and depicted in algorithmic scale (LoglO). B. Data are shown as mean +/- SD of three technical replicates. HEI-193 cells, Caco-2 and RAW 264.7 cells were infected at MOI 50, 10 and 5 respectively with 14028 parental strain, VNP20009 wt and mutant strains at 37 C for one Ih followed by gentamycin treatment. The number of invading bacteria was determined by recording CFU/ml/TO. Results are expressed as percentage of invasion (CFU/ml/TO). Each infection is the mean +/- SEM of at least 3 independent experiments. Ordinary One-way ANOVA with Dunnetf s multiple comparisons test was used to compared samples to VNP20009 control.

FIG. 5. Overexpression of SPI-1 and SPI-2 S. Typhimurium VNP20009 genes does not increase invasion to Caco-2 and RAW 264.7 cells. Caco-2 and RAW 264.7 cells were infected at MOI 10 and 5 respectively with 14028s parental strain, VNP20009 wt and overexpressing mutant strains either under Ptac promotor or PhoPQ promoter, at 37 C for one Ih followed by gentamycin treatment. The number of invading bacteria was determined by recording CFU/ml/TO. Results are expressed as percentage of invasion (CFU/ml/TO). Each infection is the mean +/- SEM of at least 2 independent experiments. Ordinary One-way ANOVA with Sidak’s multiple comparisons test was used to compared samples to VNP20009 control.

FIG. 6. Validation of SpiC-overexpressing strain in vitro conditions. Relative RNA expression of the spiC gene in VNP20009 wt, spiC and ywC'-overexpressing mutant strains grown in Mi minimal medium supplemented with either lOpM or lOmM magnesium sulfate. Fold changes were calculated relative to the rpoD housekeeping gene. The gmk gene was used as control. Each experiment is the mean +/- SEM of 2 independent experiments. 2way ANOVA with Dunnett’s multiple comparisons test was used to compared samples to VNP20009 control.

FIGs. 7A-D. Preliminary behavior data on FVB/N mice show low level of toxicity in liver and spleen in mice injected with spiC mutant strain. A. Survival rate of different doses of VNP2009, \spiC and PBS-injected mice over time. Long-rank (Mantel-Cox) test was used to compare samples (n= 3 mice/group). Body weight was monitored upon i.p. injection of PBS. VNP20009 wt and AspiC mutant strains at 2 different doses, 2xl0⁵ (B), 2xl0⁶ (C) CFU of bacteria over time. Data are shown as body weight percentage loss (n= 3 mice/group). Repeated-measured two-way ANOVA with Tukey’s test was used to compare VNP20009 and \spi(' injected mice to PBS controls. Data are shown as mean +/- SEM. Blue asterisks depict statistically significance comparison to PBS, Black asterisks comparison to VNP20009 samples. D. Liver (left panel) and spleen (right panel) tissues from VNP2009 and AspiC-injected mice, were weighted at day 11 post i.p. injection and compared to PBS- injected control mice. Graphs are showed was grams of tissue (n= 3 mice/group). Ordinary- one-way ANOVA with Tukey’s multiple comparisons test was used to compare samples. Data are shown as mean +/- SEM.

DETAILED DESCRIPTION

Bacteria-mediated cancer therapy (BCT) utilizing gram negative organisms was introduced by William Coley in the mid- 19th century when he utilized live Streptococcus pyogenes to treat solid tumors¹⁷. The rationale for bacterial cancer therapy is that some bacterial strains, including the gram-negative bacteria Salmonella Typhimurium (S. Typhimurium)¹⁸’¹⁹. can specifically home to and proliferate within hypoxic areas of angiogenic tumors inducing both direct lysis of tumor cells, as well as, establishment of antitumor immune responses²⁰. Further, bacterial injection of tumors has been shown to be anti- angiogenic²¹’²². Thus, in addition to directly inducing cancer cell death, bacteria can act as immune-oncology and anti-angiogenic agents targeting highly vascularized tumors and establishing an immune control preventing the development of new tumors.

There is a substantial body of preclinical and clinical data supporting BCT as an immunotherapeutic strategy^{23 24}, and for 4 decades intravesical application of a live attenuated strain of Mycobacterium bovis has been the only FDA-approved treatment of bladder carcinoma in situ²⁵. BCT utilizing attenuated strains ol'X. Typhimurium has demonstrated clear efficacy in several preclinical cancer models^23,26’²⁷. Early-phase clinical testing of attenuated S. Typhimurium -based BCT utilizing intravenous, direct intratumoral or oral delivery have demonstrated safety but failed to show efficacy²⁴-²⁸'³⁰. This lack of efficacy may be due to the rapid division of cancer cells, as well as, use of intravenous delivery. Bacterial inoculum is limited by toxicity with systemic delivery and in these trials lack of efficacy may have been dose related. There is currently one BCT approved by the U.S. Food and Drug Administration: a live attenuated strain of Mycobacterium bovis that for the last 4 decades has been the standard of care for high- risk non-muscle- invasive bladder cancer²⁵.

However, BCT has never been suggested as a possibility for benign neoplasms, perhaps because benign tumors tend to be immunologically cold^31,32. Thus, bacteria therapy has never been tested in the context of slow-growing benign tumors, such as schw annomas, for which traditional cancer therapies targeting mainly highly replicating cells are not effective. Provided herein is a preclinical study supporting bacterial treatment of schwannoma, a benign neoplasm of the peripheral nervous system. It w as hypothesized that intratumoral injection of atenuated S. Typhimurium could have the potential to directly kill schwannoma cells, inhibit angiogenesis, and convert the immunologic tumor microenvironment from one that is relatively ‘cold’ to ‘hot.’ It was further hypothesized that the combination of immunologic cell death (were it to occur), generation of a pro-immunogenic tumor environment, and VEGF/angiogenesis inhibition could synergize to generate an adaptive anti-tumor immune response.

To test these hypotheses, in WO2020/176764. the effects of two atenuated 5. Typhimurium strains (VNP20009 and AppGpp) were evaluated in both a xenograft human- NF2 schwannoma model in nude mice and an allograft mouse NF2-schwannoma model in syngeneic immune competent FVB/N mice. The data showed that intratumoral injection of attenuated S'. Typhimurium controlled schwannoma growth in both models. I.T. S’. Typhimurium injection of schwannoma results in tumor cell killing and, in immune competent mice, induction of a systemic anti-tumor adaptive immune response. This antitumor immune response controlled grow th of non-bacterially- injected tumors that were present at the time of bacterial treatment, as well as preventing development of “rechallenge” tumors following treatment. S’. Typhimurium increased tumor infiltrating CD4+ helper and CD8+ cytotoxic T cells, and decreased CD25+ Tregs in bacterially- injected and contralateral non-injected contralateral and rechallenge (other than the effect on CD4+ cells) allograft schwannomas, further supporting the presence of an anti-tumor adaptive immune response. Addition of systemic PD-1 immune checkpoint inhibition to i.t. S. Typhimurium injection enhanced schwannoma control of bacterially injected and contralateral non-injected, but not rechallenge, tumors. Investigation of tumor infiltrating lymphocytes (TILs) demonstrated increased numbers of CD4+ helper and CD 8+ cytotoxic T cells and decreased numbers of CD25+ regulatory T cells in schwannomas injected with atenuated S. Typhimurium.

Bacteria-mediated cancer therapy is atractive because it can selectively target and colonize the tumor microenvironment⁵¹'⁵³. The atenuated strain of Salmonella Typhimurium, VNP20009, has been so far one of the most characterized strains for bacteria-mediated cancer therapy and evaluated in several solid tumor models⁵⁴’⁵⁵. The success of VNP20009 in preclinical studies prompted phase 1 clinical trials in which VNP20009 was intravenously administered to metastatic cancer patients. This study reported that even though tumor colonization was observed, tumor regression was not achieved at the highest tolerated dose²⁴. Therefore, in this study we focused on engineering VNP20009 strain to optimize tumor specificity and biosafety. We aimed at improving specificity by generating bacteria with improved binding and invasion of schwannoma cells. Thus, we targeted the SPI-1 T3SS of S. Typhimurium, 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 the outcome of bacterial treatment of schwannoma. AsipB S. Typhimurium only partially abrogated tumor regression in the mouse syngeneic model, suggesting that host invasion by Salmonella Typhimurium is multifactorial and alteration of one single component is not sufficient to achieve the desired tumor specificity.

The tumor microenvironment, and in particular macrophages therein, plays an active role in murine modeling of S. Typhimurium for treatment of melanoma⁵⁶. In a previous study we suggested that invasion and survival of bacteria within macrophages is crucial for their anti-tumor efficacy. Furthermore, bacteria within phagocytes disseminate to systemic sites, such as spleen and liver^38,57’ ⁵⁸. Given that SPI-2 T3SS is activated within phagosome and is required for survival within macrophages, we explored the impact of the SPI-2 T3SS in schwannoma murine therapy. We deleted spiC gene from VNP20009 chromosome and assessed bacterial load and anti-tumor efficacy in tw o schwannoma murine models. The antitumor effect of AspiC was comparable to that of the wild-type VNP20009 in syngeneic and xenograft murine models, respectively. Interestingly, when bacterial load was assessed in the syngeneic model, we reported low er colonization in tumor-bearing mice treated with AspiC strain. Therefore, the AspiC strain is as efficacious as VNP20009 in treating schw annoma- bearing mice while more attenuated than VNP20009.

As in vivo toxicity remains still a concern for the clinical translation of VNP20009- mediated immunotherapy, we assessed the biosafety of the AspiC strain in FVB/N mice. We showed that mice which were systemically injected with \spi( ' showed less body weight loss, higher survival rate and less tissue injury’ in comparison with mice treated with VNP20009. After three days of injection, the fitness of AspiC in spleen and liver tissues was reduced, indicating the favorable safety profile observed in mice. Given that improvement in antitumor specificity may occur if higher doses of bacteria could be delivered, we demonstrated that \spi( ' can be safely administrated to mice at higher doses than VNP20009, which can improve anti-tumor specificity and render AspiC a better candidate for clinical applications.

Of note, Pawelek et al., have shown that deletion of SPI-2 genes in VNP20009 abrogates tumor growth suppression, suggesting that SPI-2 locus is essential for the A Typhimurium antitumor effect in a melanoma murine model⁵⁹. They also showed that deletion of several SPI-2 genes did not result in significant attenuation of virulence. This finding is in contrast with our AspiC attenuation data and with the general knowledge of the SPI-2 function in systemic spread in murine models³⁸'⁶⁰'⁶². Nonetheless, the authors did not exclude the possibility of compensatory mutations for the unexpected amplification of SPI-2 mutants within the tumor. Moreover, <S’. Typhimurium might exploit different intracellular trafficking pathways in tumor cells, suggesting that the tumor microenvironment plays a crucial role in S. Typhimurium anti-tumor activity.

The mechanism by which \spiC strain might operate within tumors is not yet fully elucidated. Uchiya et al. reported the involvement of SpiC in flagellum synthesis, suggesting that alteration of SpiC level influences Salmonella motility and therefore virulence in the Salmonellosis model⁶³. While VNP20009 is non-motile, its flagella machinery is fully expressed, and therefore it is plausible that SpiC by modulating the flagellin expression might alter its activation of host pathways and therefore immune responses. Moreover, we previously reported that VNP20009 shifts the balance of macrophages from M2 tumorigenic to Ml tumoricidal¹² Therefore, we speculate that SspiC might have a more pronounced impact on macrophage polarization. Future studies are needed to investigate the mechanism of the \spi( ' bacterial anti-tumor effect in schwannomas.

Conclusively, in the present study, we developed and assessed the antitumor efficacy and safety profiles of novel attenuated S. Typhimurium strains in preclinical schwannoma mouse models. These data support our effort of enhancing schwannoma bacterial therapy using bacterial strains with enhanced safety profile while maintaining their antitumor efficacy.

Methods of Treatment

As shown herein, a AspiC mutant strain of attenuated A Typhimurium was effective in treating schwannoma models in mice.

Schwannomas are genetically stable, slow growing, and highly vascularized with large hypoxic areas³⁴ . These features could make schwannomas an ideal homing environment for bacteria and a potentially perfect target for bacteria cytotoxic and anti- angiogenic features. In addition, the capacity of bacteria to induce immune responses allows treatment of multiple distal lesions and the establishment of control mechanisms that prevent the occurrence of new schwannomas throughout a patient’s life, a feature typical of these tumors.

The methods described herein include methods for the treatment of benign nervous system tumors, and for reducing the risk of having a benign nervous system tumor (subjects who are at risk include those who have a disorder associated with development of these tumors, including neurofibromatosis type 1 (NF1); NF2-related schwannomatosis (NF2 or NF2-SWN); schwannomatosis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF)). In some embodiments, the tumor is a schwannoma. Schwannoma tumors are composed of Schwann-lineage cells and form along peripheral, spinal and cranial nerves. These tumors can cause pain, sensory/motor dysfunction, and death through compression of peripheral nerves, the spinal cord, and/or the brain stem. Multiple schwannomas in peripheral distal and intracranial nerves are the hallmark of neurofibromatosis 1 and 2 (NF1 and NF2), and schwannomatosis, three types of nerve sheath tumors. Schwannomas are benign tumors composed of neoplastic dedifferentiated Schwann cells. Although typically nonmalignant and slow growing, these tumors can have devastating consequences for patients. They can cause extreme pain and compromise sensory/motor functions, including hearing and vision. Schwannomas in NF2 are frequently associated with neurological deficits, such as paraesthesias, weakness, or hearing loss, and similar tumors in schwannomatosis often cause excruciating pain. Some schwannomas become very large, causing compression of adjacent organs or structures, and can lead to paralysis or death due to progressive spinal cord or brainstem compression. Schwannomas may arise sporadically, without presenting any genetic features of NF1, NF2 and schwannomatosis. Most vestibular schwannomas are sporadic schwannomas, so their incidence is very significant. Vestibular schwannomas usually occur as single tumors, not as multiple tumors throughout the body. In some embodiments of any of the aspects, a subject in need of treatment for a schwannoma can be a subject having or diagnosed as having a condition selected from the group consisting of neurofibromatosis type 1 (NF1); NF2-related schwannomatosis (NF2); schwannomatosis; meningioma; nerve sheath tumor; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibrosarcoma; neurofibroma; neurofibromatosis (NF); malignant peripheral nen e sheath tumor; and a combination thereof. Subjects who can be treated using the present methods include mammals, e.g., humans and non-human veterinary subjects, e.g., cats, dogs, horses, goats, cows, and so on. The present standard of care for patients with NF2 and schwannomatosis is surgical resection or radiosurgery of symptomatic tumors to reduce tumor size. Unlike in the case of sporadic schwannomas, in which typically only a single tumor is present and surgery’ is generally an efficacious treatment strategy as long as the lesion is accessible for resection, in schwannomatosis and NF2, which present with multiple tumors, resection is confounded by both the inaccessibility of many tumors and by risk of nerve damage, including major motor dysfunction, significant sensory loss (including deafness in the case of NF2 vestibular schwannomas), and neuropathic pain. Thus, for most individuals there is substantial morbidity associated with schwannomas in both NF2 and schwannomatosis, as well as with the current therapies. This suffering and debility, in combination with the paucity of therapeutic options, makes the treatment of schwannomas a major unmet medical need.

Generally, the methods include administering a therapeutically effective amount of attenuated Salmonella, e.g., S'. Typhimurium as described herein, optionally in combination with a checkpoint inhibitor, to a subject who is in need of, or who has been determined to be in need of, such treatment. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, and intratumoral (i.t.) administration. In preferred embodiments, the i.t. route is used to maximize bacterial dose and minimize potential dose limiting toxicity (DLT). One of skill in the art would be able to identify a subject as having a benign nervous system tumor. In some embodiments, the subject is a subject having or diagnosed as having a benign tumor or tumor-associated condition selected from the group consisting of: neurofibromatosis type 1 (NF1); neurofibromatosis type 2 (NF2); schwannomatosis; 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, e.g., does not have cancer. In some embodiments, the subject has a condition associated with an increased risk of a benign nervous system tumor, e.g., neurofibromatosis type 1 (NF1); neurofibromatosis type 2 (NF2); or schwannomatosis.

The term "effective amount" as used herein refers to the amount of a composition needed to alleviate at least one or more symptom of the disease or disorder, and relates to a sufficient amount of pharmacological composition to provide the desired effect. The term "therapeutically effective amount" therefore refers to an amount of a composition that is sufficient to provide a particular anti -tumor effect when administered to a typical subject. An effective amount as used herein, in various contexts, would also include an amount sufficient to delay the development of a symptom of the disease, alter the course of a symptom disease (for example but not limited to, slow ing the progression of a symptom of the disease), or reverse a symptom of the disease. Thus, it is not generally practicable to specily an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. Administration of a therapeutically effective amount of a compound described herein for the treatment of a benign nerv ous system tumors can result in decreased tumor size, tumor number, tumor growth rate, or likelihood of recurrence, e g., after treatment with a method described herein. The present methods thus include the administration of attenuated S. Typhimurium strains to suppress tumor growth. As shown herein, to enhance efficacy of the therapy, in preferred embodiments the present methods can utilize intra-tumoral injection of bacteria, rather than intravenous delivery, which increases bacterial concentration within tumor and minimizing systemic toxicity. As shown herein, direct injection of attenuated S. Typhimurium into schwannoma had a vaccine-like action inducing an anti-tumor adaptive immune response.

Atenuated S. Typhimurium

As used herein, the term "attenuated" refers to a strain that has been rendered to be less virulent compared to the native strain, thus becoming harmless or less virulent. Attenuated does not mean inactivated. Attenuation confers decreased likelihood of pathogenicity, including septic shock, in both strains. Although the present data relates primarily to the spiC deletion mutant of strain VNP20009, other attenuated strains can also be used. Methods of generating attenuated S'. Typhimurium strains are known in the art. including directed or random mutagenesis followed by screening for reduced virulence. Directed mutation, e.g., of the aroA gene (aroA is part of the shikimate pathway connecting glycolysis to synthesis of aromatic amino acids; aroA deficient Salmonella strains are described e.g., in Feigner et al, mBio, 2016, 7: e01220-16); the gene purl (defective in purine synthesis); or the asd gene (defective in aspartate-semialdehyde dehydrogenase required for cell wall synthesis) can be used. Attenuated strains of salmonella are disclosed in WO 2014/005683; WO 2016/202459; WO 2013/09189; and US 20200038496 (attenuated S. typhi Ty21a). Strains that can be used in the present methods include attenuated versions of 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"), Salmonella enterica serovar Enteriditis ("S. enteriditis"), Salmonella enterica serovar Kentucky ("S. ken lucky"). Salmonella enterica serovar Infantis ("S. infantis"), Salmonella enterica serovar Pul lorum ("S. pullorum"), Salmonella enterica 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"), Salmonella enterica serovar Choleraesuis var. kunzendorf ("S’, cholerae kunzendorf), and Salmonella enterica serovar minnesota ("S. minnesota"). See, e.g., WO/2008/039408 and US 20200023053; US 20190153452; US 20170333490 and US 20180339032; Grant et al., PLoS Pathog. 2012 Dec; 8(12): el003070; Tennant and Levine, Vaccine. 2015 Jun 19; 33(0 3): C36-C41.

In preferred embodiments, the attenuated strains used in the present methods do not comprise Clostridium novyi (see, e.g., W02014160950). In preferred embodiments, the attenuated strains used in the present methods do not comprise a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter (see. e g., US 20170333490).

Combination Therapy

The present methods can include administration of the attenuated Salmonella strain in combination one or more other treatments. For example, the studies disclosed in W02020/176764 demonstrated that i.t. VNP20009 of schwannoma in immunocompetent mice resulted in an increase in the percentage of tumoral helper CD4+ and cytotoxic CD8+ T cells, and concomitant decrease in percentage of CD+25 Tregs. These changes in tumor infiltrating T cell populations in conjunction with a shift to Ml tumoricidal macrophages is suggestive of S. Typhimurium induced adaptive anti-tumor immune response. The high PD- L1 expression reported in schwannomas indicates resistance to cell-mediated immunity in the tumor immune microenvironment.

Given this, in WO2020/176764 the effect of addition of PD-1 immune checkpoint inhibition on efficacy of i.t. S. Typhimurium (VNP20009)- associated schwannoma growth control and development of host anti -tumor adaptive immunity was evaluated. The data showed that this combination resulted in enhanced tumor regression of bacterially injected schwannomas that was associated with an elevation in numbers of CD4+ helper and CD8+ cytotoxic T cells, a reduction in number of CD25+ regulatory T cells infiltrating both bacterially- injected and non-injected tumors.

Thus, the present methods can include administering (together or separately) a combination of bacteria together with a checkpoint inhibitor, e.g., an inhibitor of PD-1 signaling, e.g., an antibody that binds to PD-1, CD40, or PD-L1, or an inhibitor of Tim3 or Lag3, e.g., an antibody that binds to Tim3 or Lag3, or an antibody that binds to CTLA- 4. Exemplary anti -PD-1 antibodies that can be used in the methods described herein include those that bind to human PD-1 ; an exemplary PD-1 protein sequence is provided at NCBI Accession No. 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, in volumab. 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 at NCBI Accession No.

NP 001241.1, NP 690593.1, NP 001309351.1, NP_001309350.1 and NP_001289682.1.

Exemplary antibodies include those described in W02002/088186; W02007/124299;

WO2011/123489; WO2012/149356;

WO2012/111762; W02014/070934; US20130011405; US20070148163; US20040120948;

US20030165499; and US8591900, including dacetuzumab, lucatumumab, bleselumab, teneliximab, ADC-1013, CP-870,893, Chi Lob 7/4, HCD122, SGN-4, SEA-CD40, BMS- 986004, and APX005M. In some embodiments, the anti-CD40 antibody is a CD40 agonist, and not a CD40 antagonist. Exemplary CTLA-4 antibodies that can be used in the methods described herein include those that bind to human CTLA-4; exemplary CTLA-4 protein sequences are provided at NCBI Ace No. NP_005205.2. Exemplary antibodies include those described in Tarhini and Iqbal, Onco Targets Ther. 3: 15-25 (2010); Storz, MAbs. 2016 Jan; 8(1): 10-26; US2009025274; US7605238; US6984720; EP 1212422; US5811097; US5855887; US6051227; US6682736; EPl 141028; and US7741345; and include ipilimumab, Tremelimumab, and EPR1476. Exemplary anti-PD-Ll 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 at NCBI Accession No. NP_001254635.1, NP 001300958. 1, and NP 054862. 1. Exemplary antibodies are described in

US20170058033; WO2016/0 1 142A1 ; W02016/007235A1 ; WO2014/195852A1 ; and WO2013/079174A1, including 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 cellular receptor 2 or HAVCR2) antibodies that can be used in the methods described herein include those that bind to human Tim3; exemplary' Tim3 sequences are provided at NCBI Accession No. NP_116171.3. Exemplary antibodies are described in WO2016071448; US8552156; and US PGPub. Nos. 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; exemplary' Lag3 sequences are provided at NCBI Accession No. NP_002277.4. Exemplary antibodies are described in Andrews et al., Immunol Rev. 2017 Mar;276(l): 80-96; Antoni et al., Am Soc Clin Oncol Educ Book. 2016;35:e450-8; US PGPub. Nos. 20180326054; 20180251767; 20180230431; 20170334995; 20170290914; 20170101472; 20170022273; 20160303124, and include BMS-986016.

The present methods can also include administering (together or separately) a combination of bacteria together with an angiogenesis inhibitor. A number of 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); Lenvatimb mesylate (LENVIMA); Pazopanib (VOTRIENT); Ramucirumab (CYRAMZA); Regorafenib (STIVARGA); Sorafenib (NEXAVAR); Sunitinib (SUTENT); Thalidomide (SYNOVIR, THALOMID); Vandetamb (CAPRELSA); or Ziv-afhbercept (ZALTRAP). See, e.g.. Zhang et al., Exp Neurol. 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, Curr Opin Otolaryngol Head Neck Surg. 2012 Oct;20(5):372-9; Goel et al, Cold Spring Harb Perspect Med. 2012 Mar;2(3):a006486.

Alternatively or in addition, the present methods can be used in combination with surgical resection, e g., in some embodiment of any of the aspects, the attenuated salmonella strain as described herein can be administered before, concurrently with, or after surgical removal or partial removal of a neoplasm or tumor, e.g., a schwannoma. Various treatment method of the present invention may further comprise treating the subject with surgery, radiation therapy, or chemotherapy, or a combination thereof.

Pharmaceutical Compositions and Methods of Administration

The methods described herein include the use of pharmaceutical compositions comprising attenuated salmonella described herein as an active ingredient. Pharmaceutical compositions typically include a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions, e.g., checkpoint inhibitors and/or angiogenesis inhibitors, e g., as known in the art and/or discussed herein.

Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, and intratumoral administration. EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Materials and Methods

The following materials and methods were used in the Examples set forth below.

Bacteria strains

Salmonella Typhimurium VNP20009 (YS1646; ATCC Cat. BAA-3199) and A. coll strains were routinely grown aerobically at 37 °C in LB broth or on LB plates. Antibiotics were added to the medium at the following concentrations: 0.1 mg mL ¹ streptomycin and 0.1 mg/ml carbenicillin.

Generation of S. Typhimurium strains

Deletions of the spiC and sipB genes in S. Typhimurium were generated using methods previously described⁴⁷. The plasmids used are listed in Table 1 below. Briefly, for generating a gene deletion vector, ~600bp flanking regions that included 15 nucleotides of gene of interest coding sequences (CDS) were amplified and cloned into the pRE107 suicide vector using Gibson Assembly (New England Biolabs). For the generation of the spiC overexpressing strain, the phoN neutral site was in-frame deleted as above described and swapped with spiC CDS under the control of either the Tac-promoter (pTac) or the phoPQ- activated promoter (PphoPQ). 600bp flanking regions that included 15 nucleotides of phoN CDS. pTac or PphoPQ and spiC CDS were PCR amplified and cloned into pRE107 suicide vector. Genes of interest were deleted by introduction of the corresponding suicide vectors into VNP20009 via bacterial conjugation. Integration of the plasmids in the correct location on the chromosome was verified by PCR.

Table 1. Plasmids Used

* Edwards. R. A., Keller, L. H. & Schifferli, D. M. Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149-157, doi: 10.1016/s0378- 1119(97)00619-7 (1998).

Invasion assay

Invasion studies were performed as previously described⁴⁸. Briefly, overnight cultures of S. Typhimurium were diluted 100: 1 in LB and grown aerobically at 37 °C until OD600= 0.8 (mid log phase) shaking. Normalized cultures were incubated with monolayers of Caco-2, RAW274.6 macrophages or HEI-193 schwannoma cells for 1 h at 37°C in 5% CO2 at a multiplicity of infection of 5: 1, 10: 1 and 50=1. respectively. After Ih of co-incubation, cells were washed three times with PBS and incubated with media containing 50 pg/ml gentamicin for 30 minutes at 37°C in 5% CO2. After antibiotic treatment the cells were washed three times with DMEM and lysed with 1% Triton. The number of CFU per ml was calculated by plating serial dilutions of cell lysates on LB plates. qPCR

Cultured bacterial RNA was extracted as previously described⁴⁹. Briefly, overnight cultures of S. Typhimurium were diluted 100: 1 in LB and grown at 37°C until mid-log phase (OD600= 0.8-1). Bacteria w ere collected from 2 mL culture and resuspended in 1 mL TRIzol (Thermo Fisher Scientific). RNA subsequently purified using PureLink RNA mini kit (Invitrogen) and treated with Turbo DNA-free kit to remove any contaminating DNA (Ambion). RNA w as quantified by Qubit (Thermo-Fisher Scientific), and qRT-PCR was performed using Kapa SYBR Fast One-Step Universal qRT MasterMix in a thermocycler (Eppendorf Mastercycler RealPlex2). RNA levels were normalized to that of rpoD for each strain.

Motility assay

Our sw arming assay w as carried out by inoculating 3 pl of normalized overnight bacterial culture in LB medium containing 0.3% (W/V) agar and incubate at 30 °C for 12h. Spreading of bacterial growth a few centimeters from the point of inoculation was viewed as positive for motility. The assay was repeated in triplicate.

Purine auxotrophy assay

For the purine auxotrophy assay, overnight cultures of S. Typhimurium were diluted and grown aerobically at 37 °C in M9 minimal media or M9 supplemented with 20pg/ml Adenine. Optical density at 600nm (ODsoo) was measured using the Synergy plate reader. Animals

All animal experimentation was approved by and conducted under the oversight of the Massachusetts General Hospital (MGH, Boston, MA) Institutional Animal Care and Use Committee. Animals, nu/nu and FVB/N mice were kept on a 12: 12 light-to-dark cycle with ad libitum access to food and water.

Animal models

We generated schwannomas by injecting 3xl0⁴ human HEI-193FC or IxlO⁴ mouse 08031-8FC schwannoma cells directly into the left sciatic nerves of isoflurane-anesthetized athymic nude (nu/nu, 5-7-week-old males; National Cancer Institute [NCI]), or syngeneic FVB/N (5-7-week-old males; Charles River Laboratories) mice, respectively as described⁴⁴, with a glass micropipette and a gas-powered microinjector (IM-300; Narishige, Tokyo, Japan)⁵⁰. Prior to injection, we trypsinized and rinsed the cells with cold PBS, and suspended each inoculum in 0.5 pl of PBS. Two weeks after implanting HEI-193FC cells or one week after implanting 08031-9FC cells, we injected the sciatic nerve tumors with 10⁴ CFU S. Typhimurium in 2 pl PBS. We monitored tumor growth with in vivo bioluminescence imaging weekly for HEI-193FC tumors and semiweekly for 08031-9FC tumors⁵⁰. We captured images wi th a high efficiency IVIS Spectrum (Caliper Life Sciences, Hopkinton, MA) ten minutes after injecting mice intraperitoneally with the Flue substrate d-luciferin. For the immunocompetent syngeneic Sc. model, we resuspended 08031-9 cells in PBS and mixed with Matrigel (1 : 1) (BD Biosciences), then grafted 10⁵ cells Sc. in the immunocompetent mice. We estimated tumor volume from W x L x L x TI/6), where width (W) and length (L) corresponded to the two largest dimensions³⁶. Once a tumor reached 150 mm³, we injected i t. S. Typhimurium strains or PBS vehicle controls by using an insulin syringe. Excised tumors were homogenized in PBS and extracts were diluted serially and plated on Luria Broth (LB) plates. After incubating the plates overnight at 37°C S. Typhimurium colonies were counted, and bacterial load (CFU/g) determined as viable bacterial colonies cultured from tumor homogenates normalized to weight of recovered tissue 23

Biosafety animal study

VNP20009 and spiC strains were administered by intraperitoneal injection to naive FVB/N mice at various doses. Animals were monitored and weighed daily. At day 3 or day 11 post injection, organs were removed, weighed aseptically, and homogenized in 5 ml of sterile PBS. The homogenate was then plated onto modified LB media and incubated for 24 h at 37°C. The colony-forming units (cfu) per gram of tissue were determined by counting colonies and normalizing them by the weight of the recovered organ.

Statistical analysis

All group values are presented as mean ± standard error of the mean. The details of the statistical tests carried out are indicated in the respective figure legends, one-way ANOVA (between-subjects factors) or two-way ANOVA (between-subjects factors) was used followed by post hoc multiple comparisons test. Statistical analyses were performed with Prism software (GraphPad).

Example 1. Optimization of bacterial binding to and invasion of schwannoma cells

Despite solid preclinical data supporting VNP20009 as anti-tumor agent in a variety of tumor models, clinical trials of VNP20009 for metastatic malignant melanoma have been disappointing ^24,35. In this study, we aimed to boost tumor specificity by modifying endogenous loci of the Salmonella chromosome to enhance binding and invasion of schwannoma cells. In support of this hypothesis, our previous study suggested that alteration of Salmonella adhesion to the tumor cells might enhance tumor specificity¹². Hence, we targeted S. Typhimurium T3SS, the needle-like structure secreting virulence proteins directly into host cells. We generated an in-frame deletion mutant strain of sipB gene (AsipB), encoded by Salmonella pathogenicity islands (SPI) locus, to abrogate function of the SPI-1 T3SS, required for invasion of epithelial cells and inflammation. We did not observe any changes in the ( \sipB) strain growth when cultured in rich laboratory media such as Lysogeny broth (LB; Fig. 4A-B).

Next, we evaluated the antitumor activity' oiSsipB strain in a syngeneic mouse schwannoma model^12,36. We implanted mouse NF-2-schwannoma tumor cells subcutaneously (s.c.) into syngeneic FVB/N mice, followed by i.t. injection with PBS, VNP20009 or SsipB strains. Tumor growth was assessed over time via direct measurement with a caliper. Our data show ed that the \sipB strain significantly controlled tumor growth, compared to PBS control (Fig. 1 A). Tumor growth profile for \sipB and PBS-injected mice diverged 2 weeks after i.t. bacterial injection. Despite the enhanced tumor suppression in mice treated with \sipB strain compared to PBS, the greatest tumor control w as observed in mice treated with VNP20009. The effect of VNP20009 on tumor volume was evident 10 days after i.t. bacterial injection and by the end of the study VNP20009 reduced tumor growth approximately 2-fold compared to the PBS control (Fig. 1A). We then evaluated the capacity of S. Typhimurium mutant strains to bind and invade relevant mammalian cell lines, by performing a gentamycin protection assay. After applying S. Typhimurium 14028 parental strain, VNP20009, and \sipB to a monolayer of either Caco- 2 epithelial cells, RAW274.3 macrophages, or HEI-193 schwannoma tumor cells, the extracellular bacteria were removed with gentamycin. Intracellular bacteria and bacteria attached to the host cells were recovered after 1 hour to measure percentage of bacterial invasion. The data revealed that deletion of the sipB did not alter the level of cell invasion when compared to VNP20009 in any cell lines (Fig. 4B). To further investigate this finding, we overexpressed sipB protein, by replacing the phoN neutral site of the Salmonella chromosome with a construct consisting of either the Ptac inducible promoter or the phoPQ activated promoter, which is known to be active in vivo, with the sipB gene. We then assessed whether the bacterial with the altered protein profile is responsible for improved binding and invasion. As judged by the gentamycin assay, overexpression of SPI-1 encoded sipB protein did not improve the binding and/or invasion to any of the tested cell lines. Our data indicated that deletion of sipB only partially abrogated tumor efficacy in the syngeneic mouse-NF2 schwannoma model, suggesting that alteration of SPI-1 T3SS enhances tumor specificity (Fig. 5). Moreover, given the complexity of the tumor microenvironment it is not surprising that our in vitro invasion data did not correlate with our in vivo tumor control findings. Thus, also indicating that in vitro screening of Salmonella strains for tumor cell invasion cannot be used as a predictor of in vivo schwannoma growth control.

Example 2. Tumor-suppressive effects of a VNP20009 spiC deletion mutant in syngeneic mouse-NF2 and xenograft human-NF2 mouse models.

An overriding goal of our work is to develop an attenuated S. Typhimurium strain with similar or better anti-tumoral efficacy than VNP20009, but with a better toxicity profile. SpiC, one of the effectors encoded by the SPI-2 locus and a component of the T3SS, is required for secretion of the T3SS secretion proteins³⁷. Its deletion results in significantly decreased colonization of intestinal sites in a murine typhoid fever model³⁸. Therefore, we deleted spiC from the VNP20009 chromosome (terming the mutant, VNP20009-A5 ?/C), and verified that such deletion did not alter bacterial fitness in LB standard media. Similar to the sipB mutant strain, our data showed that deletion of spiC did not increase invasion of relevant mammalian cell lines, such as Caco-2. RAW274.3 HEI-193 cell lines (Fig. 4B). This finding is consistent with previous observations in which SspiC did not affect invasion but enhanced survival within the macrophages^38,39. Next, we assessed the tumor control and colonization profile of the VNP20009-s/?zC mutant in syngeneic and xenograft mouse models. First, we generated subcutaneous murine (08031-9 cell line) schwannoma tumors in syngeneic immunocompetent (FVB/N) mice. Once the tumor size reached approximately 150 mm³, we injected intratumorally PBS, VNP20009 wt, or \spi( ' mutant strains to test their therapeutic effects. As expected, i.t. injection of VNP20009 controlled tumor growth within approximately 10 days (Fig. IB). Notably, animals treated with i.t. injection of \spiC mutant strain showed significantly slower tumor growth in comparison to mice treated with PBS, and similar antitumoral efficacy as mice treated with attenuated VNP20009 wt strain (Fig. IB).

Subsequently, we sought to determine whether intratumoral treatment with SpiC overexpressing (\phoN::spiC under the PphoPQ promoter) S. Typhimurium would reverse the anti-tumoral effect of spiC mutant strain. First, we validated the expression level of SpiC for the overexpressing strain in vitro. The PhopQ activated promoter has been demonstrated to positively regulate target gene expression in mice⁴⁰. Its functionality in laboratory conditions is mimicked by low pH and low magnesium content in the media^41,42. By judged by the RNA relative level of SpiC analyzed by qPCR, we confirmed that the SpiC overexpressing strain cultured in minimal media with low- magnesium expressed more spiC transcripts than the VNP20009 control strain cultured in the same conditions (Fig. 6). Next, we assessed the antitumoral efficacy the SpiC overexpressing S. Typhimurium in subcutaneous syngeneic schwannoma mouse model. In contrast to animals that received intratumoral injections of either VNP20009 or spiC, treatment with the SpiC overexpressing S. Typhimurium abrogated tumor suppression, suggesting that fine-tuned levels of SpiC protein are necessary for clinical efficacy under the tested conditions (Fig. IB). We also evaluated bacterial burden profiles at day 2, 7 and 12 post i.t. injection by calculating colony forming units (CFU)/g of tumors. The load of Aspic strain at day 7 and 12 drastically decreased in comparison to VNP20009 and SpiC overexpressing strain, indicating that low er doses of \ piC mutant strain are required to attain efficacy levels similar to a high-dose treatment with VNP20009 strain (Fig. 1C).

To assess the broader applicability of our approach, we also examined antitumoral efficacy of the generated strains in the syngeneic and xenograft intrasciatic schwannoma murine models. We implanted firefly luciferase (Flue) expressing HEI-193FC (human-NF2 schwannoma line) and 08031-9FC (murine JVF2-defi cient schwannoma line) tumor in the sciatic nerves of athymic nude (nu/nu) immunocompromised and syngeneic immunocompetent (FVB/N) mice, respectively⁴³'⁴⁵. Once tumors size stabilized, we injected tumor-bearing sciatic nerves with approximately 10⁴ attenuated VNP20009 or \spiC mutant bacteria or PBS control. Tumor growth was assessed via in vivo bioluminescence imaging every 2 days for the syngeneic model and every week for the xenograft model. In both tumor models tumor growth profiles for VNP20009 or AspiC and PBS groups diverged as early as 7 days post i.t. treatment (Figs. ID and IF). Moreover, VNP20009 and AspiC treated mice demonstrated significant tumor suppression, as compared PBS treated ones (Figs. ID and IF). Strikingly, in the xenograft model, VNP20009 and AspiC groups resulted in undetectable tumor levels in 3/5 and 4/5 treated mice, respectively (Fig. IF). We additionally examined the bacterial burden within tumors of syngeneic-treated mice. At 22 days post-implantation, we observed approximately 2xl0⁸ CFU/g of VNP20009 wt in tumor tissues, whereas we detected 3x10⁴ CFU/g of AspiC mutant strain, corroborating our previous finding on the subcutaneous syngeneic model (Fig. IE). These data suggest that even though the AspiC mutant strain colonizes tumor environment less efficiently, it is still efficacious in treating schwannoma tumors.

Example 3. Toxicity and behavior of attenuated AspiC strain in FBV/N mice

As in vivo toxicity is still a concern for the clinical translation of VNP20009-mediated cancer therapy, we performed a biosafety⁷ study for VNP20009 and \spi(' strains. In our pilot experiment, we injected groups of FVB/N mice (N=3) intraperitoneally with PBS, 2xl0⁵ (low dose), 2xl0⁶ (medium dose), or 2xl0⁷ (high dose) CFU of VNP20009 or AspiC strains. Sickness and body weight changes were monitored daily. After 11 days, animals were sacrificed to evaluate organs inflammation by measuring spleen and liver weight. The survival rate data of mice which were injected with low or medium doses of either VNP20009 or \spiC strains was indistinguishable from that of the mice injected with PBS control (100%) (Fig. 7A). However, the highest dose of VNP20009 strain induced a significant drop-in survival rate as only 33% of mice survived by 11 days post bacterial injection. Mice injected with AspiC strain at the same dose survived until the end of the study. We calculated the LD50 of VNP20009 strain in FVB/N mice to be 2xl0⁷ (Fig. 7A). We observed no weight difference for mice injected with PBS or 2xl0⁵ CFU of either VNP20009 or AspiC strains (Fig. 7B). Of note, post 2xl0⁶ CFU injection, AspiC strain caused a temporary⁷ drop of body w eight, how ever mice regained body w eight within 24 hours. Mice injected with 2xl0⁶ CFU of VNP20009 strain dropped 10% of their body weight by the end of the observation period (Fig. 7C). To determine whether \spiC strain induces less organ inflammation in the injected mice, we collected liver and spleen from all the injected animals at the end of the study and compared tissue weight to the PBS injected controls. Tissues extracted from mice injected with either dose of spiC strain were indistinguishable from tissue collected from PBS- injected mice. By contrast, mice injected with low and medium doses of VNP20009 significantly developed tissue inflammation, as judged by the increased weight of liver and spleen (Fig. 7D). This suggests that genetic ablation of spiC induced less systemic toxicity than the wt VNP20009 strain and therefore is more attenuated in FVB/N mice.

Next, we investigated the bacterial burden in systemic sites in infected FVB/N mice. Our pilot experiment on mice inj ected with the highest dose of VNP20009 indicated that 66% of animals perished within 4 days of injection, therefore we selected day 3 post injection as an early time point. We intraperitoneally injected PBS or 2x10⁷ CFU of either VNP20009 or ^spiC strains. We monitored body weight loss and showed an initial drop of body weight within 24 hours for both groups of mice injected with S. Typhimurium strains. However, by day 3, mice injected with \spiC gained more weight than mice injected with the same dose of VNP20009 (Fig. 2A). Liver and spleen collected from mice injected with VNP20009 were highly colonized with an average of 10⁶ and 10⁷ CFU/g in liver and spleen, respectively. The load of spiC burden was significantly less than VNP20009 in both tissues, suggesting indeed that SpiC affects colonization in tumors and systemic sites (Fig. 2B). Measurement of liver and spleen weight at day 3 post injection indicated no significant differences between VNP20009 and spiC strains, suggesting that inflammation of systemic sites could occur at a later point (Figs. 2C-D).

To corroborate our finding, we replicated the biosafety study utilizing more animals per group and assessed bacterial burden at day 10 post injection (Fig. 3). PBS or two distinct doses - 2xl0⁶ and 2x10⁷ CFU- of either VNP20009 or \spiC Salmonella strains were injected i.p. in FVB/N mice and sickness monitor over time. As previously observed, i.p administration of \spi(' strain to mice at 2xl0⁶ or 2xl0⁷ CFU doses produces no mortality (Fig. 3 A). Consistent with our previous study. 2xl0⁷ CFU of VNP20009 was sufficient to kill 66% of the injected mice, confirming the LD50 previously established (Fig. 3 A). A drop of body weight of mice injected with 2x10⁶ CFU spiC was observed within 48 hours post injection, however animal gained weight by the end of the observation period (Fig. 3B). At day 10 post injection, liver and spleen were collected, and bacterial load enumerated as CFU per grams of tissue. The level of VNP20009 in spleen and liver reached ~ 2xl0³ and 2xl0⁴ CFU indicating that tissue colonization for both strains at day 10 was lower than what we observed at 3 days post infection and no significant differences were observed between VNP20009 and \spiC (Fig. 3C). Finally, we assessed tissue inflammation by measuring tissue weight and compared to healthy control tissues. As previously observed, tissues extracted from VNP20009-injected mice were heavier than the ones from Vs/vC-injected mice, confirming that \ piC is less toxic than wild-type VNP20009 (Fig. 3D). Taken altogether, our data indicated that \ piC strain colonizes systemic sites to a lesser extent and therefore is safer for treatment of schwannomas in mice.

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

Claims

WHAT IS CLAIMED IS:

1 . A method of a treating a subject having or at risk of having a nervous system tumor, the method comprising administering to the subject a therapeutically effective amount of a composition comprising a live attenuated spiC deletion variant strain of a 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 having or diagnosed as having a benign tumor or tumor-associated condition selected from the group consisting of: neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannomatosis; 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 a benign nervous system tumor.

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

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

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

8. The method of claim 7, wherein the attenuated, spiC deletion variant strain of S. typhimurium is Salmonella enterica serovar typhimurium strain VNP20009.

9. The method of claim 1, wherein the composition does not comprise Clostridium novyi.

10. The method of claim 1, wherein the attenuated Salmonella do not comprise a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter.

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

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

13. The method of claim 1 1, 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 inhibitor of VEGF is Bevacizumab.

16. A composition comprising a live attenuated spiC deletion variant strain of Salmonella enterica serovar Typhimurium strain VNP20009.

17. A composition comprising a live attenuated spiC deletion variant strain of Salmonella bacteria, optionally in combination with a checkpoint inhibitor and/or angiogenesis inhibitor, for use in a method of a treating a subject having 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 having or diagnosed as having a benign tumor or tumor-associated condition selected from the group consisting of: neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); schwannomatosis; meningioma; schwannoma: vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis (NF); or any combination thereof.

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

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

21. The composition for the use of claim 20. wherein the condition associated with an increased risk of a benign nervous system tumor is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannomatosis.

22. The composition for the use of claim 17. wherein the attenuated Salmonella is formulated to be administered intratumorally or intravenously.

23. The composition for the use of claims 17-22, wherein the attenuated Salmonella is an attenuated strain of S. typhimurium.

24. The composition for the use of claim 21, wherein the attenuated strain of S'. typhimurium is Salmonella enterica serovar typhimurium strain VNP20009.

25. The composition for the use of claim 17. wherein the composition does not comprise Clostridium novyi.

26. The composition for the use of claim 17, wherein the attenuated Salmonella do not comprise a lysis gene or cassette operably linked to an intracellularly induced Salmonella promoter.

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

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

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

30. The composition for the use of claim 17. wherein the inhibitor of VEGF is Bevacizumab.