Treatment of Benign Nervous System Tumors Using Attenuated Salmonella typhimurium

Abstract

Compositions and methods for the treatment of benign nervous system tumors, including schwannomas, using attenuated Salmonella typhimurium and optionally one or more checkpoint inhibitors.

Description

Technical Field

[0001] Claim of Priority This application claims the benefit of U.S. Patent Application No. 62 / 811,066, filed Feb. 27, 2019. The entire content of the foregoing is incorporated herein by reference.

[0002] Provided herein are compositions and methods for treating benign nervous system tumors, including schwannomas, using attenuated Salmonella typhimurium and optionally one or more checkpoint inhibitors.

Background Art

[0003] Schwannomas are slow-growing benign tumors that originate from Schwann lineage cells 1、2 Depending on location and size, these tumors can cause various neurological deficits of function gain and loss, including hearing loss, imbalance, tinnitus, movement disorders, and severe pain 3、4 and, in some cases, can lead to death due to compression of the brainstem 5 Schwannomas can occur sporadically (thus referred to as "sporadic schwannomas") or as part of the debilitating genetic syndromes neurofibromatosis type 2 (NF2) and schwannomatosis 6 Treatment of schwannomas is mainly limited to surgical resection and symptomatic management of pain. Resection, which is not curative for many patients, often involves additional neurological damage and may be unrealistic due to location or the number of tumors 7 Due to the slow replication nature of these benign lesions, anticancer therapeutics have not demonstrated efficacy against schwannomas 8、9、10 Bevacizumab is currently the only drug therapy generally accepted for schwannomas, and bevacizumab temporarily stabilizes tumor growth by targeting the highly angiogenic nature of a subset of these neoplasms 8、9、11Unfortunately, current strategies for pain management are often inadequate, and thus the disease burden is further increasing. The fact that schwannomas appear in multiple locations and new lesions occur throughout life further complicates treatment. Therefore, schwannomas and related diseases result in lifelong pain that cannot be stably controlled with current treatment options. SUMMARY OF THE INVENTION

[0004] Schwannomas are slow-growing benign neoplasms that occur throughout the body, for example along the spinal cord, within the skull. Schwannomas often first appear in childhood or adolescence and new tumors can develop throughout life. These tumors cause pain, sensory / motor dysfunction, and death due to compression of peripheral nerves, the spinal cord, and / or the brain. Coupled with a lack of treatment options, the significant pain and debilitation associated with schwannomas have made their treatment a major unmet medical need. Described herein is a treatment approach for benign neoplasms, including schwannomas, involving intratumoral (i.t.) injection of attenuated Salmonella typhimurium (S. typhimurium). Current results demonstrate the ability of this i.t. S. typhimurium to control tumor growth in both a xenograft human NF2 schwannoma model in nude mice and an allograft transgenic mouse-schwannoma model in immunocompetent animals. Growth control of schwannomas in the allograft model was associated with apoptosis of tumor cells, decreased tumor angiogenesis, and induction of an anti-tumor adaptive immune response. Intratumoral injection of S. typhimurium controlled not only the bacterially injected tumor but also simultaneously occurring distal schwannomas. Furthermore, i.t. S. typhimurium controlled the growth of rechallenge schwannomas transplanted contralateral to the primary tumor 13 days after the primary treatment. In the allograft schwannoma model, systemic application of a programmed cell death 1 receptor (PD-1) checkpoint inhibitor controlled tumor growth to a similar extent as i.t. S. typhimurium, and the combination of the two treatments resulted in an additive effect on growth control at the site of bacterial injection and a synergistic effect on the T cell subset population.

[0005] The present data support an immunotherapy that can control the growth of benign nervous system tumors, including schwannomas, and schwannoma-related neoplasms, including NF1-related tumors and meningiomas, with or without bacterial injection, by combining therapy via i.t. attenuated S. typhimurium with PD-1 checkpoint inhibition, optionally. The presented data further suggest the potential of treatment strategies for controlling the growth of tumors that occur after initial treatment. Importantly, direct injection of attenuated Salmonella typhimurium into tumors had a vaccine-like effect of inducing an anti-tumor adaptive immune response. These results represent both the first reported application of bacterial tumor therapy to benign neoplasms and the first demonstration of schwannoma immunotherapy.

[0006] Accordingly, provided herein is a method for treating a subject having or at risk of having a benign nervous system tumor. The method includes administering to the subject a therapeutically effective amount of a composition comprising live attenuated Salmonella bacteria, optionally in combination with an immune checkpoint inhibitor and / or an angiogenesis inhibitor. Also provided herein is a composition comprising live attenuated Salmonella bacteria, optionally in combination with a checkpoint inhibitor and / or an angiogenesis inhibitor, for use in a method of treating a subject having or at risk of having a benign nervous system tumor.

[0007] In some embodiments, the subject is a subject diagnosed as having or at risk of having a benign tumor or tumor-related 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 (i.e., is not 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, such as neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannomatosis.

[0008] In some embodiments, the attenuated Salmonella is administered intratumorally or intravenously.

[0009] In some embodiments, the attenuated Salmonella is the Salmonella enterica serovar typhimurium strain VNP20009, which is an attenuated strain of S. typhimurium, for example, with modified lipid A (msbB-) and a purine auxotrophic mutation (purI-).

[0010] In some embodiments, the composition does not contain Clostridium novyi.

[0011] In some embodiments, the attenuated Salmonella does not contain a lytic gene or cassette operably linked to an intracellular-inducible Salmonella promoter.

[0012] In some embodiments, the checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling, for example, an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

[0013] In some embodiments, the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR), for example, bevacizumab.

[0014] Furthermore, provided herein is a method of treating a benign schwannoma in a mammal, the method comprising administering to the mammal a therapeutically effective dose or titer of an attenuated strain of a pathogenic enterobacterium. In some embodiments, the attenuated strain of the pathogenic enterobacterium is Salmonella typhimurium. In some embodiments, the attenuated strain of Salmonella typhimurium has purl and msbB gene deletions, and the strain is named VNP20009. In some embodiments, the attenuated strain of Salmonella typhimurium has a deletion in guanosine 5'-diphosphate-3'-diphosphate synthesis, and the strain is named 8ppGpp. In some embodiments, the administration includes, but is not limited to, intravenous injection or direct injection into the benign schwannoma. In some embodiments, the schwannoma includes, but is not limited to, a neurofibroma or a schwannoma. In some embodiments, the tumor includes, but is not limited to, those associated with neurofibromatosis type 1, neurofibromatosis type 2, schwannomatosis, or sporadic schwannoma.

[0015] In some embodiments, the method comprises administering to the mammal a therapeutically effective dose of an attenuated strain of a pathogenic enterobacterium and a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor includes, but is not limited to, a peptide, an antibody, a small molecule, a microRNA, an antisense oligonucleotide, or a small interfering RNA. In some embodiments, the checkpoint inhibitor is a monoclonal antibody that binds to an epitope of an antigen. In some embodiments, the monoclonal antibody binding epitope is on a PD-1 or CTLA-4 antigen.

[0016] In some embodiments, the mammal is a human.

[0017] Also provided herein are pharmaceutical compositions comprising an attenuated strain of a pathogenic enteric bacterium in a pharmaceutically acceptable carrier and optionally a checkpoint inhibitor. In some embodiments, the attenuated strain of the pathogenic enteric bacterium is Salmonella typhimurium.

[0018] 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 CTLA-4 antigen.

[0019] 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 for use in the present invention are described herein; 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.

[0020] Other features and advantages of the present invention will become apparent from the following detailed description and figures, as well as from the claims.

Brief Description of the Drawings

[0021]

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Mode for Carrying Out the Invention

[0022] Bacterial-mediated cancer therapy (BCT) using Gram-negative organisms was introduced in the mid-19th century when William Coley used Streptococcus pyogenes to treat solid tumors. 12 。The rationale for bacterial cancer therapy is that several bacterial strains, including the Gram-negative bacterium Salmonella typhimurium (S. typhimurium) 13~21 specifically inhabit the hypoxic regions of angiogenic tumors, proliferate within those regions, and induce both direct lysis of tumor cells and the establishment of an antitumor immune response. 22 Furthermore, injection of bacteria into tumors has been shown to be anti-angiogenic. 23,24Therefore, in addition to directly inducing cancer cell death, bacteria can act as immuno-oncology and anti-angiogenic agents that target highly vascularized tumors and establish immune control to prevent the development of new tumors.

[0023] There is a body of preclinical and clinical data supporting BCT as an immunotherapy strategy 20、25、32、57、58 and, for 40 years, the intravesical application of a live attenuated strain of Mycobacterium bovis has been the only FDA-approved treatment for bladder cancer in situ. 29 BCT using an attenuated strain of S. typhimurium has demonstrated clear efficacy in several preclinical cancer models. 16、18~20 Initial-phase clinical trials of attenuated S. typhimurium-based BCT using intravenous, direct intratumoral, or oral delivery have demonstrated safety but have not been able to show efficacy. 25、26、27、28 This lack of efficacy may be due to the rapid division of cancer cells and the use of intravenous delivery. Bacterial inoculation is limited by toxicity due to systemic delivery, and in these trials, the lack of efficacy may be dose-related. Currently, there is one BCT approved by the US Food and Drug Administration: namely, a live attenuated strain of Mycobacterium bovis, which has been the standard treatment for high-risk non-muscle-invasive bladder cancer for the past 40 years. 29 .

[0024] However, perhaps because benign tumors tend to be immunologically cold, BCT has never been suggested as a possibility for benign neoplasms. 66、67Therefore, bacterial therapy has not been tested in the context of slow-growing benign tumors such as schwannomas, for which conventional cancer therapies that primarily target highly replicating cells are ineffective. Provided herein are preclinical studies supporting bacterial therapy for schwannomas, which are benign neoplasms of the peripheral nervous system. Intratumoral injection of attenuated S. typhimurium has been hypothesized to directly kill schwannoma cells, inhibit angiogenesis, and potentially convert the immunological tumor microenvironment from relatively “cold” to “hot”. Furthermore, a hypothesis has been proposed that the combination of immunological cell death (if it occurs), generation of a pro-immunogenic tumor environment, and VEGF / angiogenesis inhibition may act synergistically to generate an anti-tumor adaptive immune response.

[0025] To test these hypotheses, the effects of two attenuated S. typhimurium strains (VNP20009 and ΔppGpp) were evaluated in both a xenograft human NF2 model in nude mice and an allograft mouse - schwannoma model in syngeneic immunocompetent FVB / N mice. The data showed that the growth of schwannomas was controlled in both models by intratumoral injection of attenuated S. typhimurium. Intratumoral (i.t.) injection of S. typhimurium resulted in tumor cell death and, in immunocompetent mice, induced a systemic anti - tumor adaptive immune response. This anti - tumor immune response controlled the growth of non - bacterially injected tumors that were present at the time of bacterial treatment and prevented the development of "re - challenge" tumors after treatment. S. typhimurium increased tumor - infiltrating CD4+ helper and CD8+ cytotoxic T cells and decreased CD25+ Tregs in bacterially injected and contralateral non - injected and re - challenged (except for the effect on CD4+ cells) allograft schwannomas, which further supported 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 in bacterially injected and contralateral non - injected tumors but not in re - challenge tumors. Examination of tumor - infiltrating lymphocytes (TILs) demonstrated an increase in the numbers 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 S. typhimurium.

[0026] In this study, we tested the ability of two attenuated S. typhimurium strains, namely VNP20009 and ΔppGpp, to suppress the growth of schwannomas. Attenuation reduces the potential for pathogenicity, including septic shock, in both strains. In vitro evaluation revealed that VNP20009 is more invasive than ΔppGpp in both cultured macrophages and schwannoma cell lines, but less invasive than wild-type S. typhimurium (Figures 13A and B). Exposure of cultured macrophages to both S. typhimurium strains released inflammatory cytokines, but neither VNP20009 nor ΔppGpp induced cytokine release when co-cultured with schwannoma cell lines (Figures 14A and B and 15A and B). This suggests that the bacterium-macrophage interaction may play an important role in the observed antitumor effect of S. typhimurium.

[0027] In vivo data showed that intratumoral (i.t.) injection of either VNP20009 or ΔppGpp monotherapy regressed tumor growth in a human NF2 xenograft model, with no difference in the extent of regression between the two test strains. Furthermore, in an allograft schwannoma model in immunocompetent mice, tumor growth was controlled by i.t. injection of VNP20009. The therapeutic effect of VNP20009 was reflected by an increase in apoptotic bodies in the tumor microenvironment (Figure 1D) and an increase in the release of inflammatory cytokines such as IL-18, TNF-α, and IFN-γ (Figures 2E and F) compared to ΔppGpp or PBS. No increase in systemic cytokine levels was observed in mice injected with S. typhimurium (Figure 16). Further studies focused on the analysis of changes in the immune profile in the tumor microenvironment of an immunocompetent schwannoma model after bacterial i.t. injection of VNP20009 or PBS i.t. injection.

[0028] M2 macrophages and myeloid-derived suppressor cells (MDSCs) have been shown to infiltrate vestibular schwannomas and are associated with progressive tumor growth. 42、59 . Unlike M2 macrophages that promote tumors, M1 macrophages are immunostimulatory, inhibit tumor growth, and form an adaptive immune response at least in part through phagocytosis and antigen presentation. 60~65 . In a syngeneic schwannoma model, injection of i.t. VNP20009, which controls tumor growth, increased the ratio of M1 macrophages to M2 macrophages among CD45+F4 / 80+ tumor-activated macrophages (TAMs) by 3 days after bacterial injection.

[0029] Treatment method As shown herein, a strain of attenuated Salmonella, such as S. typhimurium, safely administered to patients with metastatic melanoma and renal cell carcinoma 25、30、31 was effective in treatment in a mouse schwannoma model. Schwannomas are genetically stable, grow slowly, and are highly vascularized in large hypoxic regions. These characteristics make schwannomas an ideal homing environment for bacteria and potentially a perfect target for the cytotoxic and anti-angiogenic characteristics of bacteria. In addition, the ability of bacteria to induce an immune response enables the establishment of control mechanisms for treating multiple distal lesions and preventing the development of new schwannomas, which is typical of these tumors, throughout the patient's life.

[0030] The methods described herein include methods for the treatment of benign nervous system tumors. In some embodiments, the tumor is a schwannoma. Schwannoma tumors are composed of Schwann lineage cells and are formed along peripheral nerves, spinal nerves, and cranial nerves. These tumors can cause pain, sensory / motor dysfunction, and death due to compression of the peripheral nerves, spinal cord, and / or brainstem. Multiple schwannomas in peripheral distal nerves and intracranial nerves are characteristic of three types of schwannoma tumors, neurofibromatosis 1 and 2 (NF1 and NF2), and schwannomatosis. A schwannoma is a benign tumor composed of neoplastic dedifferentiated Schwann cells. Typically non-malignant and slow-growing, these tumors can have devastating consequences for patients. They can cause extreme pain and can impair sensory / motor functions including hearing and vision. Schwannomas in NF2 are often associated with neurological deficits such as paresthesia, weakness, or hearing loss, and similar tumors in schwannomatosis often cause intractable pain. Some schwannomas can grow 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 showing any of the genetic characteristics of NF1, NF2, or schwannomatosis. Since most vestibular schwannomas are sporadic schwannomas, their incidence is very important. Vestibular schwannomas typically occur as a single tumor rather than 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 1 (NF1); neurofibromatosis 2 (NF2); schwannomatosis; meningioma; schwannoma tumor; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibrosarcoma; neurofibroma; neurofibromatosis (NF); malignant peripheral nerve sheath tumor; and combinations thereof. Subjects that can be treated using the methods of the invention include mammals, such as human and non-human animal subjects, such as cats, dogs, horses, goats, cows, etc.

[0031] The current standard treatment for patients with NF2 and schwannomatosis is surgical resection or radiosurgery of symptomatic tumors to reduce tumor size. Typically, only a single tumor is present, and unlike in the case of sporadic schwannomas where surgery is generally an effective treatment strategy as long as the lesion is accessible for resection, in schwannomatosis and NF2 presenting multiple tumors, resection is complicated by both the inability to access many tumors and the risk of nerve injury such as major motor dysfunction, significant sensory loss (including hearing loss in the case of NF2 vestibular schwannomas), and neuropathic pain. Thus, for most individuals, there are schwannomas in both NF2 and schwannomatosis, and substantial morbidity associated with current treatment methods. Coupled with the lack of treatment options, this pain and debilitation have made the treatment of schwannomas a major unmet medical need.

[0032] Generally, the method involves administering a therapeutically effective amount of attenuated Salmonella, such as S. typhimurium described herein, optionally in combination with a checkpoint inhibitor, to a subject in need of or determined to be in need of such treatment. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, and intratumoral (i.t.) administration. In a preferred embodiment, the i.t. route is used to maximize the dose of bacteria and minimize potential dose-limiting toxicity (DLT). One of ordinary skill in the art will be able to identify a subject as having a benign nervous system tumor. In some embodiments, the subject is 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, having or diagnosed as having a benign tumor or tumor-related condition. 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 benign nervous system tumors, such as neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannomatosis.

[0033] As used herein, the term "effective amount" refers to the amount of a composition necessary to alleviate at least one or more symptoms of a disease or disorder, and relates to a pharmacological composition in an amount sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to the amount of a composition sufficient to provide a particular anti-tumor effect when administered to a typical subject. The effective amount as used herein also includes an amount sufficient to delay the onset of symptoms of the disease, alter the course of symptoms of the disease (e.g., but not limited to, slow the progression of symptoms of the disease), or reverse the symptoms of the disease in various situations. Thus, it is generally not practicable to specify an exact "effective amount". However, for any given case, the 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 the compounds described herein for the treatment of benign nervous system tumors can, for example, result in a decrease in tumor size, number of tumors, tumor growth rate, or likelihood of recurrence after treatment by the methods described herein.

[0034] Accordingly, the method of the present invention comprises administration of an attenuated S. typhimurium strain for suppressing tumor growth. As shown herein, to enhance the effectiveness of the treatment, in a preferred embodiment, the method can utilize intratumoral injection of bacteria that increases the bacterial concentration within the tumor and minimizes systemic toxicity rather than intravenous delivery. As shown herein, direct injection of attenuated S. typhimurium into a schwannoma has a vaccine-like effect and induces an anti-tumor adaptive immune response.

[0035] attenuated S. typhimurium As used herein, the term "attenuated" refers to a strain that has been made less virulent compared to the natural strain, and thus is harmless or has reduced virulence. Attenuation does not mean inactivation. Attenuation reduces the potential for pathogenicity, including septic shock, in both strains. Current data mainly relate to VNP20009 and ΔppGpp, but other attenuated strains can also be used. Methods for generating attenuated Salmonella strains are known in the art, such as directed or random mutagenesis followed by screening for reduced virulence. For example, directed mutations in the aroA gene (aroA is part of the shikimate pathway that connects glycolysis to the synthesis of aromatic amino acids; aroA-deficient Salmonella strains are described, for example, in Feigner et al, mBio, 2016, 7: e01220-16); the purl gene (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 pamphlet; WO 2016 / 202459 pamphlet; WO 2013 / 09189 pamphlet; and US Patent Application Publication No. 20200038496 (attenuated S. typhi Ty21a). Strains that can be used in current methods include 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 B), Salmonella enterica serovar Paratyphi C (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. kentucky), Salmonella enterica serovar Infantis (S. infantis), Salmonella enterica serovar Pullorum (S. pullorum), Salmonella enterica serovar Gallinarum (S. gallinarum), Salmonella enterica serovar Muenchen (S. muenchen), Salmonella enterica serovar Anaturn (S. anatum), Salmonella enterica serovar Dublin (S. dublin), Salmonella enterica serovar Derby (S. derby), Salmonella enterica serovar Choleraesuis var.attenuated versions of (S. cholerae kunzendorf), and Salmonella enterica serovar minnesota (S. minnesota). See, for example, WO 2008 / 039408 and US 20200023053; US 20190153452; US 20170333490 and US 20180339032; Grant et al., PLoS Pathog. 2012 Dec; 8(12): e1003070; Tennant and Levine, Vaccine. 2015 Jun 19; 33(0 3): C36-C41.

[0036] In preferred embodiments, the attenuated strain used in the method is free of Clostridium novyi (see, for example, WO 2014160950). In preferred embodiments, the attenuated strain used in the method does not contain a lysis gene or cassette operably linked to an intracellular inducible Salmonella promoter (see, for example, US 20170333490).

[0037] Combination therapy The method of the present invention can include administration of an attenuated Salmonella strain in combination with one or more other treatments. For example, current studies have demonstrated that i.t. VNP20009 in schwannomas in immunocompetent mice resulted in an increase in the percentage of tumor helper CD4+ and cytotoxic CD8+ T cells, and a concomitant decrease in the percentage of CD25+ Tregs. These changes in the tumor-infiltrating T cell population associated with a shift to M1 antitumor macrophages suggest an antitumor adaptive immune response induced by S. typhimurium. The reported high PD-L1 expression in schwannomas indicates resistance to cellular immunity in the tumor-mediated immune microenvironment. 49 Taking this into account, the effect of adding PD-1 immune checkpoint inhibition on i.t. S. typhimurium (VNP20009)-associated schwannoma growth control and the development of host antitumor adaptive immunity was evaluated. The data showed that this combination resulted in enhanced tumor regression of bacterially injected schwannomas, associated with an increase in the number of CD4+ helper and CD8+ cytotoxic T cells infiltrating both bacterially injected tumors and non-injected tumors, and a decrease in the number of CD25+ regulatory T cells (Figures 3 and 4). Compared to bacterially injected tumors, the combination of VNP20009 and anti-PD-1 mAb resulted in the same enhancing effect on the T cell population (increase in CD4+ and CD8+, decrease in CD25+) in contralateral non-bacterially injected schwannomas (Figures 3D, E) and rechallenge schwannomas (Figures 4D, E), but there was no difference in growth inhibition between VNP20009 / anti-PD-1 mAb and VNP20009 alone. On the other hand, the effects of both i.t. VNP20009 and the combination of VNP20009 / anti-PD-1 mAb on schwannoma growth control appear to be greater in rechallenge tumors (Figure 4D) than in primary bacterially injected tumors (Figure 4B). The differences in the biology of bacterially injected and non-injected contralateral and rechallenge schwannomas that explain these observations have not yet been elucidated.

[0038] Accordingly, the methods of the invention can include administering, together or separately, 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, in combination with a bacterium.

[0039] 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 by NCBI accession number NP_005009.2. Exemplary antibodies include those described in U.S. Patent No. 8,008,449; U.S. Patent No. 9,073,994; and U.S. Patent Application Publication No. 2011 / 0271358, and include PF-06801591, AMP-224, BGB-A317, BI754091, JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, avelumab, pidilizumab, and atezolizumab.

[0040] 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 by 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 International Publication No. WO 2002 / 088186; International Publication No. WO 2007 / 124299; International Publication No. WO 2011 / 123489; International Publication No. WO 2012 / 149356; International Publication No. WO 2012 / 111762; International Publication No. WO 2014 / 070934; U.S. Patent Application Publication No. 20130011405; U.S. Patent Application Publication No. 20070148163; U.S. Patent Application Publication No. 20040120948; U.S. Patent Application Publication No. 20030165499; and U.S. Patent No. 8,591,900, and include dacetuzumab, lucatumumab, breselumab, 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.

[0041] Exemplary anti-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 by NCBI accession number 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; US Patent Application Publication No. 2009025274; US Patent No. 7605238; US Patent No. 6984720; European Patent No. 1212422; US Patent No. 5811097; US Patent No. 5855887; US Patent Provisional Application No. 6051227; US Patent No. 6682736; European Patent No. 1141028; and US Patent No. 7741345; and include ipilimumab, tremelimumab, and EPR1476.

[0042] 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 by NCBI accession numbers NP_001254635.1, NP_001300958.1, and NP_054862.1. Exemplary antibodies include those described in US Patent Application Publication No. 20170058033; International Publication No. 2016 / 061142 pamphlet; International Publication No. 2016 / 007235 pamphlet; International Publication No. 2014 / 195852 pamphlet; and International Publication No. 2013 / 079174 pamphlet, and include BMS-936559 (MDX-1105), FAZ053, KN035, atezolizumab (Tecentriq, MPDL3280A), avelumab (Bavencio), and durvalumab (Imfinzi, MEDI-4736).

[0043] Exemplary anti-Tim3 antibodies (also known as hepatitis A virus cellular receptor 2 or HAVCR2) that can be used in the methods described herein include those that bind to human Tim3; an exemplary Tim3 sequence is provided by NCBI accession number NP_116171.3. Exemplary antibodies include those described in International Publication No. WO 2016 / 071448; U.S. Patent No. 8,552,156; and U.S. Patent Application Publication Nos. 2018 / 0298097; 2018 / 0251549; 2018 / 0230431; 2018 / 0072804; 2018 / 0016336; 2017 / 0313783; 2017 / 0114135; 2016 / 0257758; 2016 / 0257749; 2015 / 0086574; and 2013 / 0022623, and include LY3321367, DCB-8, MBG453, and TSR-022.

[0044] 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 by NCBI accession number NP_002277.4. Exemplary antibodies include those 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; U.S. Patent Application Publication Nos. 2018 / 0326054; 2018 / 0251767; 2018 / 0230431; 2017 / 0334995; 2017 / 0290914; 2017 / 0101472; 2017 / 0022273; 2016 / 0303124, and include BMS-986016.

[0045] The method of the present invention may also include administering (together or separately) a combination of an angiogenesis inhibitor and bacteria. 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); everolimus (AFINITOR); lenalidomide (REVLIMID); lenvatinib mesylate (LENVIMA); pazopanib (VOTRIENT); ramucirumab (CYRAMZA); regorafenib (STIVARGA); sorafenib (NEXAVAR); sunitinib (SUTENT); thalidomide (SYNOVIR, THALOMID); vandetanib (CAPRELSA); or aflibercept (Ziv-aflibercept) (ZALTRAP). See, for example, 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.

[0046] Alternatively or in addition, the method of the present invention can be used in combination with surgical resection. For example, in some embodiments of any of the aspects, the attenuated Salmonella strain described herein can be administered before, simultaneously with, or after the surgical removal or partial removal of a neoplasm or tumor, such as a schwannoma. The various treatment methods of the present invention may further include treating the subject with surgery, radiation therapy, or chemotherapy, or a combination thereof.

[0047] Pharmaceutical Composition and Method of Administration The methods described herein include the use of a pharmaceutical composition comprising attenuated Salmonella as an active ingredient. The pharmaceutical composition typically comprises a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" includes physiological saline, solvents, dispersion media, etc. that are compatible with pharmaceutical administration. Auxiliary active compounds can also be incorporated into the composition, for example, as known in the art and / or as discussed herein, such as checkpoint inhibitors and / or angiogenesis inhibitors.

[0048] The pharmaceutical composition is typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, and intratumoral administration.

Example

[0049] The present invention is further described in the following examples, which do not limit the scope of the present invention described in the claims.

[0050] Materials and Methods In the examples described below, the following materials and methods were used.

[0051] Cell Culture The HEI-193 human schwannoma cell line (from D.J. Lim of the House Ear Institute, Los Angeles, CA) was established from schwannomas of NF2 patients, immortalized with the human papillomavirus E6 / E7 gene, and grown as described. 68、69 The mouse 08031-9 schwannoma cells (from Dr. Marco Giovannini of the Univ. of California, Los Angeles (Los Angeles), CA) were grown as described. 50The cell lines were infected with lentiviruses encoding Fluc (firefly luciferase) and mCherry for bioluminescence imaging and IHC, respectively. 70 Human MPNST (STS.26T) cells were kindly provided by Dr. David Largaespada (Masonic Cancer Center, University of Minnesota) and grown as described. 56 Human NF-1-related MPNST (S462TY) cells were kindly provided by Dr. Timothy P. Cripe (Division of Pediatric Oncology, Nationwide Children's Hospital). Human Ben-Men-1 and CH-157 cells were kindly provided by Dr. Long-Sheng Chang (Division of Pediatric Oncology, Nationwide Children's Hospital) and Dr. G. Yancey Gillespie (University of Alabama at Birmingham), respectively. To differentiate human macrophages, phorbol-12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) was added to human monocytes at a final concentration of 100 μM. After 24 h, the PMA-supplemented medium was removed, the cells were washed with PBS, and left to stand for an additional 24 h in untreated PMA-free medium to obtain macrophage phenotypic characteristics. 71 Mouse RAW macrophages were obtained (from ATCC, USA). Macrophages were cultured in RPMI medium according to the manufacturer's instructions. Before use in experiments, all cell lines were confirmed to be free of mycoplasma contamination.

[0052] Bacterial culture The attenuated Salmonella enterica serovar Typhimurium strain VNP20009 (with modified lipid A (msbB-), purine auxotrophic mutation (purI-)) was purchased (from ATCC, USA, catalog number 14028), and the ΔppGpp strain (deletion of ppGpp synthesis (RelA::cat, SpoT::kan)) was kindly provided by Dr. Karsten Tedin (Institut for Microbiology and Epizootics, Centre for Infection Medicine, Berlin, Germany). Bacterial cells were cultured overnight at 37°C, 300 rpm in Luria-Bertani (LB) broth medium containing low sodium (Difco Laboratories, USA) under aerobic conditions as described above. 31、47 Briefly, cells were grown to late logarithmic phase (OD600nm = 0.8), harvested by centrifugation at 5000 rpm for 10 minutes, washed twice with sterile 1× phosphate-buffered saline (PBS), and then used for injection into tumors or infection of cultured cells.

[0053] Animals All animal experiments were approved and conducted under the supervision of the Institutional Animal Care and Use Committee (IACUC protocol number 2014N000211) at Massachusetts General Hospital (MGH, Boston, MA). Male mice, 5 - 7 weeks old, nu / nu and FVB / N (Charles River Laboratories), were housed with free access to food and water on a 12:12 light / dark cycle and received daily health checks by the staff / veterinarians at the MGH Center for Comparative Medicine.

[0054] Animal model and intratumoral bacterial injection Sciatic nerve sheath tumors were generated as described by directly injecting HEI-193FC human or 08031-8FC mouse schwannoma cells into the left sciatic nerve of isoflurane-anesthetized mice. 72。HEI-193FC or 08031-9FC cells were trypsinized, rinsed with cold PBS, and 30,000 cells (or 10,000 for 08031-9FC) in 0.5 μl of PBS were injected into the sciatic nerves of nude athymic mice (nu / nu, 5- to 7-week-old males, National Cancer Institute [NCI]) or syngeneic FVB / N mice (5- to 7-week-old males, Charles River Laboratories), respectively, using a glass micropipette and a gas-driven microinjector (IM-300; Narishige, Tokyo, Japan). At 2 weeks after HEI-193 tumor cell transplantation or 1 week after 08031-9 tumor cell transplantation, the position of the sciatic nerve where the tumor cells were transplanted was targeted, and 10 4 CFU attenuated S. typhimurium (VNP20009 or ΔppGpp) was injected. Tumor growth was monitored in vivo by bioluminescence imaging weekly for HEI-193 and twice a week for 08031-9 as described 72 。Briefly, mice were injected intraperitoneally with the Fluc substrate d-luciferin, and 10 minutes later, signals were acquired using a high-efficiency IVIS Spectrum (Caliper Life Sciences, Hopkinton, MA). In the case of the immunocompetent subcutaneous model; 08031-9 was resuspended in DMEM and mixed with Matrigel (BD bioscience) (1:1) and then transplanted subcutaneously (s.c.) into FVB / N syngeneic mice. In the case of the NF-1 or meningioma xenograft model, human S462TY, STS.26T, Ben-Men-1 or CH-157 cells were mixed with Matrigel (1:1) and transplanted subcutaneously into nu / nu mice as described 56 。Tumor volume was estimated using the formula (W × L × L × π / 6), where width (W) and length (L) are the two largest diameters 50 。When the tumor reached 150 mm 3 in size, treatment with intraperitoneal (i.p.) PD-1 mAb (250 μg / injection, Bio X Cell, USA) was initiated and repeated every 3 days as described above 51。VNP20009 injection (10 4 CFU / 100 μl PBS) was performed intratumorally using an insulin syringe. Control group mice were treated with PBS vehicle or isotype antibody on the same schedule.

[0055] Histological and immunohistochemical analysis Animals were finally anesthetized with isoflurane (3%) and sacrificed by decapitation. As described, tumor tissues were removed and snap-frozen for hematoxylin and eosin (H&E) and immunohistochemical staining 73The tumor was maintained in OCT blocks at -80°C. Sections were stained with H&E according to a standard protocol. Proliferation marker staining was performed using an antibody against Ki67 (Abcam, Cambridge, MA). Antibodies against CD45, CD68, and CD31 were utilized for staining of leukocytes, macrophages, and angiogenesis, respectively. All antibodies were purchased from (Abcam, Cambridge, MA). Briefly, sections were air-dried overnight at room temperature (RT). Sections were fixed with pre-cooled acetone at 4°C for 10 minutes, allowed to dry, and then stained immediately. Sections were washed with PBS, blocked with a serum-free protein block (Dako, Carpinteria, CA), and peroxidase was quenched with a dual endogenous enzyme block (Dako). After washing the sections with PBS, they were incubated with the primary antibody for 1 hour at room temperature, then washed with PBS and incubated with a horseradish peroxidase-conjugated secondary antibody for 30 minutes at room temperature (RT). Sections were washed with PBS and incubated with a DAB solution (Dako). Counterstaining was achieved by immersing the sections in ethanol and xylene, mounting with Cytoseal (Richard Allan Scientific, San Diego, CA), and covering with a coverslip for visualization under a microscope. In vivo apoptosis staining was evaluated using the TACS2 TdT-DAB in situ apoptosis detection kit (Trevigen, Gaithersburg, MD). According to the manufacturer's instructions, 15-μm sections of untreated frozen nerves (cryostat) mounted on slides were fixed with 3.7% formaldehyde and stained with diaminobenzidine (DAB) for visualization under an optical microscope. The antibodies used are provided in Table 1.

[0056]

Table 1

[0057] Measurement of cytokine RNA in tumors using real-time quantitative (qRT-PCR) On the third day after bacterial injection, the tumor tissues were excised, and RNA was extracted using Trizol. Total RNA was transcribed into cDNA using SuperScript™ IV VILO™ with ezDNase enzyme (Invitrogen). Next, the samples were incubated at 37 °C for 10 minutes to digest DNA, followed by incubation at 25 °C for 10 minutes, 50 °C and 85 °C for 5 minutes in a (ProFlex PCR system, Applied Biosystems, USA). In the qPCR reaction, 20 ng of cDNA / well was used as input, and Taq-man probes were used to determine the expression of target genes. The qPCR assay was performed using the Mx3000P qPCR system (Agilent Technologies, USA) under standard cycling mode conditions. Melting curve analysis was performed using MxPro qPCR software to verify the primer efficiency of each plate and exclude non-specific amplification. The difference in cycle threshold (Ct) values between the target gene and the reference gene 18S (ΔCt) was converted to relative expression using the 2-ΔΔCT method, and the fold change was calculated by comparing the samples. All reactions were performed three times.

[0058] Measurement of cytokine proteins in tumors using ELISA On the third day after bacterial injection, the tumor tissues were excised, homogenized in NP40 lysis buffer containing protease inhibitor, and the supernatant was collected by centrifugation at 13,000 rpm for 10 minutes. According to the manufacturer's instructions, cytokine levels were measured using individual Quantikine ELISA kits (R&D systems, Minneapolis) for human and mouse: interferon gamma (IFN-γ) (BD bioscience), TNF-α (BD bioscience), IL-1β / IL-1F2 (BD bioscience), and IL-18 (BD bioscience). After measuring the color reaction of the substrate at 450 nm with the correction wavelength set to 540 nm or 570 nm using a microplate reader (SpectraMax, Molecular Devices), the results were quantified using a calibration curve.

[0059] In vitro invasiveness assay Macrophage (human THP-1 and mouse RAW264.7 macrophages) and schwannoma (human HEI-193 and mouse 08031-9) cells were cultured at 10 per well in 24-well tissue culture plates. 4 The cells were grown to a density of 100 μg / ml. The cells were washed with warm PBS and supplemented with antibiotic-free 10% FBS medium. In parallel, bacterial cells were grown to late logarithmic phase as described above and diluted with cell culture medium to represent a multiplicity of infection (MOI) of 50:1 bacteria / cell. Cultured macrophages and schwannoma cells were added with the bacteria-containing medium and placed in a 37°C incubator for 60 minutes. To determine the invasiveness of bacterial strains, the cultured cells were washed with PBS and incubated in medium containing gentamicin sulfate (50 μg / mL) for 30 minutes to kill extracellular bacteria attached to the cell surface. Next, the cells were rinsed five times with 1–2 mL of PBS, followed by the addition of 0.2 mL of 0.1% Triton X-100 for 10 minutes to lyse the cells and detach the attached bacteria. Next, LB broth (0.8 mL) was added, and each sample was vigorously mixed to prepare a homogenous suspension for serial dilutions. Ten-fold dilutions were prepared, plated on LB agar medium, and incubated overnight at 37°C to count colony-forming units (CFU).

[0060] Flow cytometry Tissues were harvested from mice (n = 3 / group), and cells were dissociated using freshly prepared lysis buffer (125 U / mL collagenase type XI, 60 U / mL hyaluronidase type I, 60 U / mL DNase 1, and 450 U / L collagenase type I (Sigma-Aldrich) in PBS containing 20 mM Hepes) in a water bath at 37°C for 1 hour, with gentle flicking every 10 minutes for proper homogenization and cell dissociation. The cell suspension was passed through a pre-wetted strainer, a 70 μm cell strainer (BD-Falcon). Cells were quantified by mixing 10 μL of the suspension with 10 μL of trypan blue and then loaded into a hemocytometer. The cell suspension was centrifuged at 2000 rpm for 10 minutes at 4°C to remove the lysis buffer, washed, resuspended in 1x PBS, and eluted at 10°C.6 The cells were maintained at [[[Cell number]]] / 100 μL. The cells were incubated with 2 μL of Fc blocking agent (BD Biosciences) for 15 minutes at room temperature. The cells were washed with PBS and then incubated with fluorescently labeled antibodies against cell surface markers or various immune markers for 1 hour in the dark, followed by a washing step with PBS and permeabilized overnight with 2% paraformaldehyde (PFA solution). The following antibodies against mouse immune markers were used for surface staining: CD45, F4 / 80, CD206, CD86, LY6G, NK1.1, NKp46, CD11b, CD11c, CD4, CD8, CD3, CD25. FACS and analysis were performed using FACSAria and LSR Fortessa with FACSDiva software (BD Bioscience) and FlowJo software. The antibodies used are provided in Supplementary Table 1.

[0061] Data analysis All data are presented as group mean ± standard error of the mean (SEM). The data were analyzed using GraphPad Prism and Microsoft Excel. Repeated measures analysis of variance (ANOVA) was utilized to compare tumor volume and / or signal as described. 74 One-way ANOVA was used to analyze cytokine expression and flow cytometry data. A P < 0.05 was considered significant.

[0062] [Example 1] Intratumoral injection of attenuated S. typhimurium suppresses tumor growth in xenograft human and syngeneic mouse schwannoma models It was evaluated whether intratumoral (it) injection of S. typhimurium could control the growth of human (HEI-193 cell line) and mouse (08031-9 cell line) schwannomas that develop in the sciatic nerves of nu / nu immunodeficient and FVB / N immunocompetent mice, respectively. Two different strains of S. typhimurium, namely VNP20009 and ΔppGpp, were evaluated. Both strains are attenuated mutant forms of wild-type bacteria that have shown higher tumor tropism and an improved safety profile in preclinical studies 19、32、33 and, for VNP20009, in clinical trials 25、34 as well.

[0063] Tumor burden was evaluated via in vivo bioluminescence imaging of firefly luciferase (Fluc) expressed by HEI-193FC (human NF2 schwannoma) and 08031-9FC (mouse NF2-deficient schwannoma) cells. Once the tumor signal had stabilized (approximately 2 weeks or 1 week after tumor implantation of HEI-193FC or 08031-9FC cells, respectively; Figures 1A and B), it was visualized directly and the tumor-bearing sciatic nerves were injected with attenuated S. typhimurium (VNP20009 or ΔppGpp) or PBS (control) (n = 8 mice / group). Tumor growth was followed for an additional 5 weeks in nude mice bearing human NF2 schwannomas and for 2 weeks in immunocompetent FVB / N mice bearing mouse NF2 schwannomas. In the xenograft schwannoma model, the study was terminated 7 weeks after tumor cell implantation - at this point, most of the bacteria-injected tumors had no bioluminescence signal. The end of the study in the intraneural syngraft model was determined by the onset of motor dysfunction in the tumor-bearing hind limb of control mice (evaluated by both our group and the animal technicians, all of whom were blinded to the study groups). Two additional replicates of this study were performed in the xenograft model and one replicate was performed in the syngraft model. (n = 8 mice / group for all studies; Figures 7A and B).

[0064] Intratumoral injection of the attenuated S. typhimurium strain VNP20009 resulted in a decrease in the bioluminescent tumor signal compared to the PBS control in all three replicates in the xenograft human NF2 schwannoma model (p < 0.01, Figure 1A; Figure 7A) and both replicates in the syngeneic mouse schwannoma model (p < 0.05, Figure 1B; Figure 7B). The growth curves of the bacterial and PBS-injected mice began to diverge in both tumor models as early as 1 week after VNP20009 injection. Bacterial treatment caused regression of the HEI-193FC tumor signal to undetectable levels in 5 out of 8 mice by 2 weeks after bacterial injection (Figure 1A). Across all three replicates of this experiment, 75% (18 / 24 mice) of the VNP20009-injected animals had no detectable tumor signal by the end of the experiment (Figure 1A, Figure 7A).

[0065] Complete regression of the tumor signal after bacterial injection was not observed in the syngeneic mouse-schwannoma model, but tumor growth control persisted until sacrifice, at which time the bioluminescent signal was approximately one-eighth in the VNP20009-injected mice compared to the PBS control (Figure 1B, Figure 7B).

[0066] Next, we tested whether the ΔppGpp strain of S. typhimurium had a similar therapeutic effect to VNP20009. In the xenograft model, the effects of VNP20009 and ΔppGpp on the tumor signal were indistinguishable from each other, but in the syngeneic mouse schwannoma model, VNP20009, but not the ΔppGpp strain of S. typhimurium, was controlling tumor growth (p < 0.05 VNP20009 vs PBS, Figure 1B), and indeed, there was a significant difference between the two strains (p < 0.05 VNP20009 vs ΔppGpp, Figure 1B).

[0067] At the end of the experiment (i.e., 5 weeks after bacterial injection in the xenograft human schwannoma model and 2 weeks after bacterial injection in the syngeneic mouse schwannoma model), histological analysis of the tumor-bearing nerves (n = 3 mice / group) collected showed abundant apoptotic bodies in both VNP20009- and ΔppGpp-injected tumors compared to PBS-injected tumors (Figures 1C and D). Quantification of tissues from the xenograft model revealed that both VNP20009-injected schwannomas (570 ± 77; p < 0.0005) and ΔppGpp-injected schwannomas (230 ± 68, p < 0.005) had more apoptotic bodies than PBS-injected tumors (4 ± 1) (Figure 1C). Comparing the apoptotic bodies induced by the two bacterial strains, the number of apoptotic cells in VNP20009-treated tumors was higher than in ΔppGpp-treated tumors of HEI-193 schwannomas (p < 0.01, Figure 1C). In xenograft human NF-2 schwannomas, there were approximately three times more apoptotic cells after VNP2009 injection than after ΔppGpp injection. In the syngeneic mouse schwannoma model, a consistent difference between groups was observed; VNP20009-injected tumors (340 ± 63) had more apoptotic cells compared to ΔppGpp-injected tumors (110 ± 27, p < 0.01) and PBS-injected tumors (6 ± 0.3, p < 0.001). ΔppGpp-injected tumors had significantly more apoptotic cells than the PBS control (p < 0.01, Figure 1D). In this model, i.t. VNP20009 injection resulted in approximately three times more apoptotic cells than ΔppGpp injection (p < 0.01, Figure 1D).

[0068] [Example 2] i.t. injection of attenuated S. typhimurium results in increased immunogenic cytokines and changes in immune cell infiltration in a syngeneic mouse-schwannoma model One of the most stimulating properties of BCT is its ability to induce anti-tumor adaptive immunity 35~37. Based on this discovery, we hypothesized that infection of schwannomas with attenuated S. typhimurium would have a vaccination effect of inducing anti-tumor adaptive immunity in the host. Immunotherapy for schwannomas is particularly valuable considering that affected individuals typically have multiple tumors, develop new tumors throughout their lives, have tumors in locations where they cannot be surgically removed without a substantial risk of major nerve damage, complete resection is often not achievable, and as mentioned above, multiple surgeries are required for tumors to develop throughout life.

[0069] Intrathecal (i.t.) injection of bacteria into xenograft and allograft schwannoma models causes apoptotic cell death (Figure 1), but we also wanted to investigate whether there was evidence of pyroptosis and / or immunogenic cell death. Therefore, injecting VNP20009 and ΔppGpp i.t. into human HEI-193 and mouse 08031-9 schwannomas growing within the sciatic nerves of nude and immunocompetent mice, respectively, may induce a broad range of indicators of the host's innate and adaptive immune responses. Lymphocyte common antigen marker CD45 38 and monocyte and tissue macrophage marker CD68 39 were used to evaluate tumor-infiltrating immune cells through analysis. Tumor-infiltrating immune cells were evaluated by immunohistochemical staining of tumors collected at 5 weeks after bacterial injection in the xenograft human NF2 model and at 2 weeks after bacterial injection in the allograft mouse schwannoma model (n = 3 tumors / treatment / model). The sacrifice time was chosen for the reasons mentioned above, namely, the dissipation of the tumor signal in most mice (xenograft model) or prior to severe morbidity in control mice (allograft model).

[0070] In the xenograft model, histological analysis of the tumor-bearing nerves revealed abundant tumor infiltration of CD45+ leukocytes and CD68+ macrophages compared to PBS-injected tumors that showed no expression of either class of cells (Figure 8). The same analysis of intraneural syngeneic mouse schwannomas showed that either i.t. injection of VNP20009 or ΔppGpp resulted in an increase in CD45+ leukocyte and CD68+ macrophage infiltration compared to PBS-injected tumors (Figure 2A). Quantification of CD45+ and CD68+ cells in the tumor microenvironment of the syngeneic model showed that this bacterial injection increased tumor-infiltrating leukocytes and macrophages compared to PBS-injected controls (Figure 2B).

[0071] Focusing on characterizing the killing of schwannomas by S. typhimurium using only syngeneic models in which these tumors develop in immunocompetent host mice, we investigated whether there are indicators of immunogenic cell responses and immunogenic cell death. Macrophages can be classified as M1 tumoricidal and M2 tumorigenic, (M1) 40 promoting the host's anti-tumor adaptive immunity or (M2) 41 subsequently inhibiting the host's anti-tumor adaptive immunity, and the balance between M1 and M2 macrophages is an important determinant of the host's anti-tumor immunity. Human schwannomas have been reported to be composed of up to 50% macrophages by cell number 42 and the higher the macrophage content, the higher the tumor growth rate 43. The macrophage population was evaluated by flow cytometry of CD86+ (for M1 type, tumoricidal) and CD206+ (for M2 type, tumorigenic) expression in CD45+F4 / 80+ cells collected from sciatic nerve allograft mouse schwannomas 3 and 7 days after i.t. injection of attenuated S. typhimurium. By i.t. injection of both VNP20009 and ΔppGpp, the macrophage balance shifted to the M1 type on day 3 after bacterial injection, and the ratio of M1 macrophages to M2 macrophages (M1 / M2) increased compared to PBS injection (p < 0.05, Figure 2C). Seven days after i.t. bacterial injection, the M1 / M2 ratio further shifted to M1 in VNP20009-injected tumors compared to PBS-treated tumors (p < 0.01, Figure 2C), while in ΔppGpp-injected tumors, the M1 / M2 ratio decreased compared to the 3-day time point and there was no longer a difference from PBS (Figure 2C). Interestingly, there was a systemic effect of i.t. injection of attenuated S. typhimurium on macrophage numbers. Over 3 days after i.t. bacterial injection, splenic macrophages (CD45+F4 / 80+) increased in the VNP20009 group (38.8%, p < 0.01) and the ΔppGpp group (32.4%, p < 0.01) compared to PBS (15.4%) (Figure 9).

[0072] In view of the increased tumor-infiltrating lymphocytes observed in bacterial-injected syngeneic schwannomas and the shift towards M1 macrophages, we investigated whether the intratumoral T cell composition was altered by intratumoral (i.t.) injection of attenuated S. typhimurium. Tumor-infiltrating helper T cells (CD3 / CD4), cytotoxic T cells (CD3 / CD8), and regulatory T cells (Tregs, CD4 / CD25) were evaluated using multicolor flow cytometry 7 days after i.t. bacterial injection (n = 3 / group). No effect on CD4+ helper T cells was observed following i.t. bacterial injection, but the percentage of CD8+ cytotoxic T cells increased following i.t. injection of either VNP20009 or ΔppGpp compared to PBS injection (7.56%, 7.56%, and 2.79% respectively, Figure 2D). Furthermore, the number of tumor-infiltrating CD25+ Tregs decreased following i.t. injection of VNP2009 or ΔppGpp compared to PBS (4.15%, 3.24%, and 8.32% respectively, Figure 2D). In all cases, these percentages represent the proportion of CD45+ cells.

[0073] Next, we investigated the effect of i.t. injection of VNP2009 or ΔppGpp on two important immunostimulatory cytokines, tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ), which are known to be involved in ICD and to regulate the survival, proliferation, and differentiation of both immune and tumor cells. 44、45 Partially, i.t. attenuated S. typhimurium elicits a shift towards M1 macrophages, so we hypothesized that bacterial infection of schwannomas induces the production of these cytokines (Figure 2C). Furthermore, S. typhimurium is involved in the processing and maturation of two pro-inflammatory cytokines, IL-1β and IL-18 19、47、48 that are known to have anti-tumor activity, including the inflammasomes NLRP3 and NLRC4 46It is a known inducer. On the 3rd day after i.t. bacterial injection, the changes in the M1 / M2 ratio mediated by VNP20009 and ΔppGpp were evident (Figure 2C), and an increase in multiple cytokines within the allograft neuroma in the sciatic nerve was observed. mRNA and proteins were extracted from the tumors, and the cytokine profile was evaluated using RT-PCR and ELISA (N = 3 / group). The transcriptional levels of the pro-inflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-18 were upregulated in the tumors injected with VNP20009 and ΔppGpp S. typhimurium compared to the control (Figure 2E). Notably, the tumors injected with VNP20009 showed higher TNF-α and IFN-γ mRNA expression levels compared to ΔppGpp (p < 0.01 and p < 0.001, respectively). As observed at the transcriptional level, the protein levels of TNF-α, IFN-γ, IL-1β, and IL-18 were elevated in the tumors injected with VNP20009 and ΔppGpp compared to the PBS control (Figure 2F). Furthermore, i.t. injection of VNP20009 resulted in more IL-18, IFN-γ, and TNF-α proteins in the injected tumors compared to ΔppGpp (p < 0.05, p < 0.01, p < 0.05, respectively; Figure 2F). Treatment with VNP2009 also led to a greater increase in NLRC4 and NLRP3 mRNA compared to ΔppGpp or PBS (p < 0.01, Figure 2E).

[0074] [Example 3] i.t. injection of S. typhimurium VNP20009 controls the growth of bacterial injection and contralateral non-injected allograft mouse neuromas. A subset of neuromas has been shown to contain CD4+ and CD8+ T cells expressing PD-1, indicating a decrease in anti-tumor immunity in these cells 49. Intratumoral (i.t.) injection of VNP20009 in allogeneic schwannomas was found to be associated with an increase in the number of CD8+ cytotoxic T cells and a decrease in the number of CD25+ Tregs, suggesting activation of the adaptive immune response. Therefore, it was evaluated that the combination of systemic anti-PD-1 monoclonal antibody (mAb) and i.t. VNP20009 injection might enhance the host's bacteria-induced anti-tumor adaptive immune response. 08031-9 mouse schwannoma cells were subcutaneously transplanted into the bilateral flanks of FVB / N mice and divided into four groups (Figure 3A shows the experimental design): i) i.t. VNP20009 (left flank tumor), ii) i.p. anti-PD-1-mAb P, iii) i.t. VNP20009 and i.p. anti-PD-1-mAb, and iv) i.t. PBS (left tumor). Subcutaneous transplantation was used instead of intrasciatic nerve transplantation because the former allows for longer survival and thus a greater opportunity for the adaptive immune response to occur. When the average tumor size reached approximately 150 mm 3 50 (on the 11th day after transplantation), VNP20009 (10 4 CFU in 100 μl) or PBS was directly injected only into the tumors transplanted on the left flank. In parallel, anti-PD-1 mAb (250 μg / injection) was injected i.p. on days 10, 13, 16, and 19 after tumor cell transplantation 51 . Monotherapy with either VNP20009 or anti-PD-1 mAb suppressed the growth of both tumors (Figure 3B) compared to i.t. PBS injection, and it was observed that i.t. VNP2009 provided greater growth control of the non-injected tumors than PD-1-mAb (p < 0.05, Figure 3B, D). The combination of i.t. VNP20009 and anti-PD-1 mAb enhanced 1) the growth control of the bacteria-injected tumors compared to either VNP20009 or anti-PD-1 mAb alone (p < 0.05, Figure 3B), and 2) the tumor growth control of the non-injected contralateral tumors compared to anti-PD-1 mAb treatment (p < 0.05), but there was no difference compared to VNP20009-treated mice (Figure 3D).

[0075] These effects on schwannoma growth suggest that i.t. VNP20009 generates an adaptive immune response capable of controlling tumor growth, and this effect may be enhanced by immune checkpoint inhibition. Considering this, the T cell subsets of these tumors were analyzed via flow cytometry, specifically helper (CD3 / CD4) T cells, cytotoxic (CD3 / CD8) T cells, and regulatory T (CD4 / CD25) T cells (Figure 3C). In the left flank tumors (Figure 3B, C), i.t. VNP20009 and systemic anti-PD-1 mAb resulted in 1) an increase in CD8+ cytotoxic T cells (11.8% and 10.8% respectively) compared to PBS injection (1.01%), 2) an increase in CD4+ helper T cells (VNP20009 (3.43%); anti-PD-1 mAb (4.21%)) compared to PBS injection (0.77%), and 3) a decrease in regulatory T cells (VNP20009 (15.5%); anti-PD-1 mAb (19.3%)) compared to PBS (31.2%). The combination of VNP20009 and anti-PD-1 mAb resulted in an additive increase in CD8+ (24.9%) and CD4+ (29.6%) T cells compared to each monotherapy and PBS (Figure 3C). There was also an additive effect of the combination of bacteria and immune checkpoint inhibition on Treg reduction (7.59%) compared to each monotherapy (VNP20009 (15.5%); anti-PD-1 mAb (19.3%), and PBS (31.2%)).

[0076] To further evaluate whether these manipulations induced a systemic host anti-tumor immune response, the same T cell population in the right flank tumor that did not receive bacterial injection was analyzed. i.t. VNP20009 (in the left flank tumor) in combination with systemic anti-PD-1 mAb resulted in a synergistic effect on infiltrating CD4+ helper T cells (23.4%) in non-injected tumors compared to VNP20009 alone (4.55%) or anti-PD-1 mAb alone (3.26%); neither monotherapy was different from PBS (2.24%) (Figure 3E). The percentage of CD8+ cytotoxic T cells in the right flank tumor was increased by either the VNP20009 / anti-PD-1 mAb combination (47.5%) or VNP20009 (41.9%) compared to both anti-PD-1 mAb (6.05%) and PBS treatment (4.92%), and the VNP20009 / anti-PD-1 mAb combination was higher than that of VNP20009 alone (Figure 3E). Finally, as shown in Figure 3E, each treatment regimen decreased the percentage of tumor-infiltrating CD25+ Tregs at the following values compared to i.t. PBS injection (in the contralateral left flank tumor): VNP20009 / anti-PD-1 mAb (14.2%), VNP20009 (22.2%), anti-PD-1 mAb (31.4%), and PBS (50.5%). Notably, the effect on Treg reduction was greatest in VNP20009 / anti-PD-1 mAb mice, and VNP20009 had a greater effect than anti-PD-1 mAb.

[0077] [Example 4] i.t. injection of Salmonella typhimurium (VNP20009) into syngeneic mouse primary schwannomas suppresses the growth of non-bacterial-injected rechallenge schwannomas. To investigate whether a sustained anti-tumor adaptive immune response can be generated by i.t. VNP20009 alone or in combination with anti-PD-1 mAb, a rechallenge model was utilized. 08031-9 mouse schwannoma cells were transplanted into the left flank of FVB / N mice and divided into the following groups as schematically shown in Figure 5A: i) i.t. VNP20009, ii) i.p. anti-PD-1-mAb P, iii) i.t. VNP20009 and i.p. anti-PD-1 mAb, and iv) i.t. PBS. When the mean tumor size reached 150 mm 3 50 (on day 8 post-transplantation), i.t. VNP20009 (10 4 CFU in 100 μl) was directly injected. Anti-PD-1 mAb (250 μg / injection) 51 was administered i.p. on days 7, 10, 13, and 16 post-tumor cell transplantation. Replicating the results shown in Figure 3, all treatment regimens, namely VNP20009 / anti-PD-1 mAb, VNP20009, and anti-PD-1 mAb, suppressed tumor growth compared to PBS, and there was an additive effect by combining VNP20009 and anti-PD-1 mAb (Figure 4B). Twelve days after bacterial injection of s.c. tumors (and 13 days after the first application of the immune checkpoint inhibitor), animals were rechallenged by transplanting 08031-9FC schwannoma cells into the contralateral sciatic nerve. Since these syngeneic transplanted schwannomas develop more rapidly intraneurally than subcutaneously due to being autologous and biased to confirm the effect, the sites within the sciatic nerve were selected for both. Intrasciatic nerve tumor growth was monitored via bioluminescence imaging, and it was revealed that tumor growth was inhibited in mice previously treated with VNP20009 or VNP20009 / anti-PD-1 mAb compared to the PBS control, and there was no difference between these two treatments (Figure 4D). There was no change in tumor growth compared to PBS with previous treatment with anti-PD-1 mAb alone (Figure 4D). Notably, the magnitude of the inhibitory effect of i.t. VNP20009 appears to be greater in the rechallenge tumors (Figure 4D) than in the primary bacterial injection schwannomas (Figure 4B).

[0078] We re-analyzed the T cell composition of the injection tumors and rechallenge tumors at the time of sacrifice by flow cytometry. In subcutaneous primary tumors, the percentage of infiltrating CD4+ helper T cells was increased by VNP20009 (8.47%), anti-PD-1 mAb (3.39%), and the combination of anti-PD-1 mAb and VNP20009 (9.39%) compared to the PBS injection control (0.99%); VNP20009 had a greater effect than anti-PD1-mAb, but when checkpoint inhibition was added to the bacteria, there was no increase in the effect compared to bacteria alone (Figure 4C). The percentage of CD8+ cytotoxic T cells in subcutaneous tumors was also increased by VNP20009 (11.2%), anti-PD-1 mAb (6.69%), and the combination of VNP20009 and anti-PD-1 mAb (15.5%) compared to PBS injection (3.83%); in this case, the increase was greater in the bacteria-injected tumors than in the immune checkpoint-treated tumors, and the combination therapy induced more CD8+ T cells than bacteria alone (Figure 4C). In contrast, the proportion of CD25+ regulatory T cells in subcutaneous tumors did not change with systemic anti-PD-1 mAb (11.2%) compared to the PBS control (12.2%), but was decreased by both i.t. VNP20009 injection (9.52%) and the combination of bacteria injection and checkpoint inhibition (4.90%; Figure 4C). This inhibitory effect of the combination therapy on tumor-infiltrating Tregs was greater than the effect of bacteria treatment alone (Figure 4C).

[0079] Quantification of the T lymphocyte population in recurrent tumors within the sciatic nerve revealed a pattern of treatment effect different from that of the primary subcutaneous tumor. The percentage of CD4+ helper T cells increased only in the tumors of mice previously exposed to the combination of VNP20009 and anti-PD-1 mAb (13%) compared to PBS control animals (5.59%); there was no effect of previous VNP20009 (7.49%) or anti-PD-1 mAb monotherapy (6.93%) (Figure 4E). In these recurrent tumors, the percentage of CD8+ cytotoxic T cells increased in mice previously treated with VNP20009 (14.1%) or the combination of VNP20009 / anti-PD-1 mAb (23.1%) compared to PBS controls (11.5%); there was no effect of previous checkpoint inhibition alone (anti-PD-1 mAb, 12.2%; Figure 4E). Similarly, the percentage of CD25+ Tregs in recurrent tumors decreased with previous treatment with VNP20009 (6.01%) or the combination of VNP20009 / anti-PD-1 mAb (3.82%) compared to PBS control mice (10.4%), but not with anti-PD-1 mAb (8.16%) (Figure 4E). There was no effect of previous immune checkpoint inhibition alone on the percentage of Tregs in recurrent schwannomas, but adding anti-PD-1 mAb to VNP20009 enhanced the inhibitory effect of previous bacterial treatment alone (Figure 4E).

[0080] [Example 5] Angiogenesis of allograft murine schwannomas is inhibited by i.t.S. typhimurium injection S. typhimurium reduces the expression of vascular endothelial growth factor (VEGF), an important angiogenesis-promoting factor 52 and has been shown to have anti-angiogenic properties in preclinical cancer models 24、53 Bevacizumab, an anti-angiogenic monoclonal antibody against VEGF-A, can control schwannoma growth in a subset of individuals with schwannomas 54In view of these observations, the effect of intratumoral (i.t.) injection of attenuated S. typhimurium on tumor vascular distribution was investigated.

[0081] The intraneural mouse 08031-9 schwannoma of the sciatic nerve was evaluated two weeks after i.t. injection of attenuated S. typhimurium, which demonstrated inhibition of tumor angiogenesis compared to the PBS injection control. Tumor angiogenesis was evaluated by direct visualization of the vascular endothelial marker CD31+ and immunohistochemistry 55 (Figure 5). Gross evaluation of the tumors (N = 6 / mouse group) showed an easily distinguishable difference between all PBS-injected schwannomas with bright red and prominent external vascular distribution compared to S. typhimurium-injected tumors that were pale in color and had minimal or no external angiogenesis (Figure 5). The number of CD31+ cells was decreased in both VNP20009-injected tumors and ΔppGpp-injected tumors compared to PBS-injected tumors (45.71 ± 4.75; Figure 5, N = 3 / group) (7.71 ± 1.89, P < 0.001 and 8.41 ± 1.68, P < 0.001, respectively).

[0082] [Example 6] Growth of xenograft human NF1, human sporadic MPNST, and human meningioma subcutaneous tumors is inhibited by i.t. S. typhimurium injection. Human NF1-related (S462TY, Figure 6A) or sporadic (STS26, Figure 6B) malignant peripheral nerve sheath tumor cells (MPNST) were subcutaneously transplanted into the left flank of immunodeficient nu / nu mice 56The effect of i.t. injection of S. typhimurium (VNP20009 and ΔppGpp) on the growth of human NF-1 xenograft models was also evaluated. Furthermore, the efficacy of i.t. S. typhimurium was demonstrated in a subcutaneous xenograft model in which nu / nu mice were transplanted with a benign meningioma (Ben-Men-1, Figure 6C) or a malignant meningioma (CH-157 MN, Figure 6D) cell line. In the test model, mice were divided into three groups (n = 5): VNP20009 or ΔppGpp (10 4 CFU) in 100 μl versus PBS injection control. When the tumor mass became visible to the naked eye, intratumoral injection was performed and tumor growth was monitored by caliper measurement.

[0083] In the NF-1 xenograft model, our data showed that i.t. injection with either VNP20009 or ΔppGpp significantly inhibited tumor growth of NF-1-related S462TY MPNST cells (P < 0.001, Figure 6A) and controlled tumor growth of rapidly growing sporadic STS26T MPNST cells (P < 0.001, Figure 6B) compared to i.t. PBS injection. At the time of sacrifice (day 31 for S462TY and day 30 for STS26T), and compared to the PBS control, VNP20009- and ΔppGpp-injected mice showed approximately one-seventh and one-fourth of the tumor size in the S462TY and STS26T models, respectively.

[0084] Similarly, in the meningioma xenograft model, i.t. injection of either VNP20009 or ΔppGpp significantly regressed tumor growth of benign (Ben-Men-1, Figure 6C) meningiomas and controlled tumor growth of malignant (CH-157, Figure 6D) meningiomas compared to the PBS control. In the benign model, on the day of sacrifice (day 38), 2 out of 5 mice showed complete regression in mice injected with either VNP20009 S. typhimurium or ΔppGpp S. typhimurium. On the day of sacrifice of malignant meningiomas, the tumor size was approximately one-third in VNP20009- and ΔppGpp-injected mice compared to the PBS control. The experiment was terminated on day 23 when the tumors in the control group ulcerated / necroticated.

[0085] References

[0086]

Table 2-1

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[0088]

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[0089]

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[0090]

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[0093]

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[0094]

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[0095] Other Embodiments Although the present invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to illustrate and not limit the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. Various embodiments of the present invention are shown below. 1. A method of treating a subject having or at risk of having a benign nervous system tumor, the method comprising administering to the subject a therapeutically effective amount of a composition comprising live attenuated Salmonella bacteria, optionally in combination with an immune checkpoint inhibitor and / or an angiogenesis inhibitor. 2. The method of item 1 above, wherein the subject is diagnosed with or has a benign tumor or tumor-related 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. 3. The method of item 1 above, wherein the subject does not have a malignant solid tumor. 4. The method of item 1 above, wherein the subject has a condition associated with an increased risk of a benign nervous system tumor. 5. The method of item 4 above, wherein the condition associated with an increased risk of a benign nervous system tumor is neurofibromatosis type 1 (NF1); neurofibromatosis type 2 (NF2); or schwannomatosis. 6. The method of item 1 above, wherein the attenuated Salmonella is administered intratumorally or intravenously. 7. The method of any one of items 1 to 6 above, wherein the attenuated Salmonella is an attenuated strain of S. typhimurium. 8. The method of item 7 above, wherein the attenuated strain of S. typhimurium is Salmonella enterica serovar typhimurium strain VNP20009, which has modified lipid A (msbB-) and a purine auxotrophic mutation (purI-). 9. The method of item 1 above, wherein the composition does not contain Clostridium novyi. 10. The method of item 1 above, wherein the attenuated Salmonella does not contain a lysis gene or cassette operably linked to an intracellular inducible Salmonella promoter. 11. The method according to 1 above, wherein the checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling. 12. The method according to 11 above, wherein the inhibitor of PD-1 signaling is an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4. 13. The method according to 1 above, wherein the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR). 14. The method according to 11 above, wherein the inhibitor of VEGF is bevacizumab. 15. A composition comprising attenuated Salmonella bacteria, optionally in combination with a checkpoint inhibitor and / or an angiogenesis inhibitor, for use in a method of treating a subject having or at risk of having a benign nervous system tumor. 16. The composition for use according to 15 above, wherein the subject has or is diagnosed with a benign tumor or tumor-related 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. 17. The composition for use according to 15 above, wherein the subject does not have a malignant solid tumor. 18. The composition for use according to 15 above, wherein the subject has a condition associated with an increased risk of a benign nervous system tumor. 19. The composition for use according to 18 above, wherein the condition associated with an increased risk of a benign nervous system tumor is neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or schwannomatosis. 20. The composition for use according to 15 above, wherein the attenuated Salmonella is formulated to be administered intratumorally or intravenously. 21. The composition for use according to any of 15 to 20 above, wherein the attenuated Salmonella is an attenuated strain of S. typhimurium. The composition for use according to item 21 above, wherein the attenuated strain of S. typhimurium is the Salmonella enterica serovar typhimurium strain VNP20009, which is accompanied by modified lipid A (msbB-) and a purine auxotrophic mutation (purI-). The composition for use according to item 15 above, wherein the composition does not contain Clostridium novyi. The composition for use according to item 15 above, wherein the attenuated Salmonella does not contain a lytic gene or cassette operably linked to an intracellular inducible Salmonella promoter. The composition for use according to item 15 above, wherein the checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling. The composition for use according to item 25 above, wherein the inhibitor of PD-1 or CTLA-4 signaling is an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4. The composition for use according to item 15 above, wherein the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR). The composition for use according to item 15 above, wherein the inhibitor of VEGF is bevacizumab.

Claims

1. A composition comprising attenuated Salmonella bacteria for use in a method of treating a subject having a benign nervous system tumor, wherein the benign nervous system tumor is a meningioma or a schwannoma, said composition.

2. The composition according to claim 1, wherein the subject does not have a malignant solid tumor.

3. The composition according to claim 1, wherein the attenuated Salmonella is formulated to be administered intratumorally or intravenously.

4. The composition according to any one of claims 1 to 3, wherein the attenuated Salmonella is an attenuated strain of S. typhimurium.

5. The composition according to claim 4, wherein the attenuated strain of S. typhimurium is Salmonella enterica serovar typhimurium strain VNP20009, which is accompanied by modified lipid A (msbB-) and purine auxotrophic mutation (purI-).

6. The composition according to claim 1, wherein the composition does not contain Clostridium novyi.

7. The composition according to claim 1, wherein the attenuated Salmonella does not contain a lysis gene or cassette operably linked to an intracellular inducible Salmonella promoter.

8. The composition according to claim 1, wherein the composition comprises the live attenuated Salmonella bacteria in combination with an antibody that binds to PD-1, CD40, PD-L1, or CTLA-4.

9. The composition according to claim 1 or 8, wherein the composition comprises the live attenuated Salmonella bacteria in combination with an inhibitor of vascular endothelial growth factor (VEGF) or its receptor (VEGFR).

10. The composition according to claim 9, wherein the inhibitor of VEGF is bevacizumab.

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