Treatment of benign nervous system tumors using attenuated salmonella typhimurium

Abstract

To provide a novel treatment method for benign nervous system tumors, including schwannomas; and to provide a composition for use in the treatment method.SOLUTION: Provided herein are methods for a treating a subject having or at risk of having a benign nervous system tumor. The methods include 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 angiogenesis inhibitor. Preferably, the attenuated Salmonella is an attenuated strain of S. typhimurium, e.g., Salmonella enterica serovar typhimurium strain VNP20009 with modified lipid A (msbB-) and purine auxotrophic mutation (purI-).SELECTED DRAWING: None

Description

[Technical Field]

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

[0002] Provided herein are attenuated Salmonella typhimurium strains. schwannoma using rium) and optionally one or more checkpoint inhibitors. and a method for treating benign nervous system tumors comprising: [Background technology]

[0003] Schwannoma is a type of tumor that grows in the Schwann lineage cells. 1、2 It is a slow-growing benign tumor derived from the Depending on their size and size, these tumors can cause hearing loss, imbalance, tinnitus, movement disorders, and severe pain. It can cause a variety of gain-of-function and loss-of-function neurological deficits, including pain. 3、4 In some cases, compression of the brainstem can lead to death 5 Schwannomas are sporadic They may occur spontaneously (hence the term "sporadic schwannomas") or may have debilitating It occurs as part of a genetic syndrome called neurofibromatosis type 2 (NF2) and schwannomatosis. may occur 6 Treatment of schwannomas primarily involves surgical removal and symptomatic management of pain. Resection is not curative for many patients and often requires additional neurological may be impractical due to location or number of tumors. 7 .these Due to the slow replicating nature of these benign lesions, anti-cancer drugs have not demonstrated efficacy against schwannomas. Not testifying 8、9、10 Bevacizumab is now generally accepted for schwannoma. bevacizumab is the only approved drug therapy for a highly selective subset of these neoplasms. Temporarily stabilizing tumor growth by targeting vascularized properties 8、9 、11 Unfortunately, current strategies for pain management are often inadequate and This further increases the burden of disease. Schwannomas can occur in multiple locations and new ones can form throughout a person's life. Treatment is further complicated by the fact that large lesions occur. Schwannoma and its associated diseases cannot be stably controlled with current treatment options. This will result in unbearable, lifelong pain. Summary of the Invention

[0004] Schwannomas are slow-growing tumors that occur throughout the body, for example, along the spinal cord and within the skull. Schwannomas are benign neoplasms that often first appear during childhood or adolescence and persist throughout life. These tumors can cause pressure on the peripheral nerves, spinal cord, and / or brain. This leads to pain, sensory / motor dysfunction, and death. Coupled with a lack of treatment options, Therefore, the significant pain and debilitation associated with schwannomas makes their treatment a major obstacle. Described herein are attenuated Salmonella Salmonella typhimurium (S. typhimurium) A therapeutic approach for benign neoplasms, including schwannomas, involves intratumoral (it) injection of The current results demonstrate the efficacy of immunoadaptation in a xenograft human NF2 schwannoma model in nude mice. This study demonstrated that the allogeneic gene expression in mice controls tumor growth in both schwannoma and allograft models. It shows the ability of S. typhimurium to The growth control of schwannoma in humans is due to tumor cell apoptosis, decreased tumor angiogenesis, and and was associated with the induction of anti-tumor adaptive immune responses. By injecting himurium, not only the bacterial tumor but also the distal schwannoma that occurs at the same time can be treated. Furthermore, S. typhimurium was shown to be effective in controlling tumors. The growth of rechallenged schwannomas implanted contralateral to the primary tumor 13 days after primary treatment was controlled. In an allograft schwannoma model, programmed cell death receptor 1 (PD-1) checkpoint inhibitors were detected. Systemic application of inhibitors prevents the development of S. typhimurium and The combination of the two treatments controlled tumor growth to a degree comparable to that seen in the case of bacterial injection. There was an additive effect on proliferation control and a synergistic effect on T cell subset populations.

[0005] Current data suggest that attenuated S. typhimurium-mediated therapy , optionally in combination with PD-1 checkpoint inhibition, in combination with bacterial injection and non-injection , benign nervous system tumors, including schwannomas, and schwannomas, including NF1-associated tumors and meningiomas The data presented support immunotherapy as a potential treatment for controlling the growth of associated neoplasms. Furthermore, this suggests a potential therapeutic strategy for controlling tumor growth that occurs after initial treatment. Importantly, attenuated Salmonella typhimurium When injected directly into tumors, it had a vaccine-like effect in inducing anti-tumor adaptive immune responses. Our results represent the first reported application of bacterial tumor therapy to benign neoplasms and the first reported application of immunotherapy for schwannoma. represents both the first demonstration of

[0006] Accordingly, provided herein are methods for treating or at risk of having a benign nervous system tumor. The method is a method for treating a subject with a cancer. The method optionally comprises administering an immune checkpoint inhibitor to a subject. Live attenuated Salmonella in combination with anti-inflammatory and / or anti-angiogenic agents - Patent Application 20070122999 The method comprises administering to a subject a therapeutically effective amount of a composition comprising the bacteria. or for use in a method of treating a subject at risk of having, optionally checked Live attenuated Salmonella ( Also provided herein are compositions comprising the Salmonella bacteria.

[0007] In some embodiments, the subject has neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF 2); Schwannomatosis; Meningioma; Schwannoma; Vestibular Schwannoma; Sporadic Schwannoma; Nerve Sclerosis fibroma; neurofibromatosis (NF); or any combination thereof A subject has or has been diagnosed as having a benign tumor or tumor-related condition. In this embodiment, 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 is diagnosed with a disorder associated with an increased risk of benign nervous system tumors. conditions associated with neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); or I have eosinophilia.

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

[0009] In some embodiments, the attenuated Salmonella is S. typhimurium ( Attenuated strains of S. typhimurium, e.g., modified lipid A (msbB-) and purine auxotrophs Salmonella enterica serovar Typhimurium ( The strain is Salmonella enterica serovar Typhimurium VNP20009.

[0010] In some embodiments, the composition comprises Clostridium novyi ) is not included.

[0011] In some embodiments, the attenuated Salmonella is an intracellular-induced Salmonella containing a lysis gene or cassette operably linked to a Salmonella promoter. do not have.

[0012] In some embodiments, the checkpoint inhibitor is a PD-1 or CTLA-4 inhibitor. inhibitors of signal transduction, such as PD-1, CD40, PD-L1, or CTLA-4 It is an antibody that binds.

[0013] In some embodiments, the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) or and inhibitors of the VEGFR receptor, such as bevacizumab.

[0014] Further provided herein are methods for treating benign nerve sheath tumors in a mammal. administering to said mammal a therapeutically effective dose or potency of an attenuated strain of a pathogenic enterobacterium In some embodiments, the method comprises administering to the subject an attenuated strain of pathogenic Enterobacteriaceae. is Salmonella typhimurium. In this state, attenuated strains of Salmonella typhimurium are The strain has deletions of the rl and msbB genes and is designated VNP20009. In some embodiments, Salmonella typhimurium The attenuated strain of is defective in guanosine 5'-diphosphate-3'-diphosphate synthesis, In some embodiments, administration is by intravenous injection or These include, but are not limited to, direct injection into benign nerve sheath tumors. In embodiments, nerve sheath tumors include, but are not limited to, neurofibromas or schwannomas. In some embodiments, the tumor is a type of tumor associated with neurofibromatosis type 1, neurofibromatosis type 2, Schwannoma. These include, but are not limited to, those associated with schwannoma, or sporadic schwannomas.

[0015] In some embodiments, the method comprises administering a therapeutically effective dose of an attenuated strain of pathogenic Enterobacteriaceae and In some embodiments, the method comprises administering a checkpoint inhibitor to the mammal. Checkpoint inhibitors include peptides, antibodies, small molecules, microRNAs, and antisense oligonucleotides. These include, but are not limited to, small interfering oligonucleotides, or 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 P in the D-1 or CTLA-4 antigens.

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

[0017] Attenuated strains of pathogenic Enterobacteriaceae in a pharmaceutically acceptable carrier, and optionally checked Also provided herein are pharmaceutical compositions comprising the POI inhibitors. In this study, attenuated strains of pathogenic enterobacteria, such as Salmonella typhimurium, murium).

[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. Binds to tope.

[0019] Unless otherwise defined, all technical and scientific terms used herein are defined by the have the same meaning as commonly understood by a person skilled in the art to which the invention pertains Methods and materials for use in the present invention are described herein; Other suitable methods and materials described in the literature may also be used. Materials, methods, and examples are provided in the It is for illustrative purposes only and is not intended to be limiting. Publications, patent applications, patents, sequences, database entries, and other references are the property of their respective owners. In case of conflict, the present specification, including definitions, will control. do.

[0020] Other features and advantages of the invention are set forth in the following detailed description and figures, as well as in the claims. It will become clear from [Brief explanation of the drawings]

[0021] [Figure 1] Intratumoral injection of attenuated S. typhimurium (S. typhimurium) inhibits schwannoma development in the human HEI-193 xenograft and murine 08031-9 allograft schwannoma models. A) Following intratumoral injection two weeks after tumor cell implantation, VNP20009 and ΔppGpp resulted in significant tumor regression (n = 8 mice / group). B) One week after tumor cell implantation, injection of VNP20009, but not ΔppGpp, resulted in growth control (n = 8 mice / group). Intratumoral VNP20009 and ΔppGpp injections increased apoptosis in both the HEI-193 xenograft model (C) and the 08031-9 allograft model (D) compared with PBS-injected controls (n = 3 mice / group; yellow arrows indicate representative apoptotic bodies). Repeated-measures ANOVA was used to compare tumor signals between groups, and one-way ANOVA was used for apoptotic body analysis. Data are presented as mean ± SEM. *p<0.05, **p<0.01. ***p<0.001. [Figure 2]S. typhimurium injection of intrasciatic nerve allograft schwannoma tumors increased proinflammatory cytokines and altered immune cell infiltration. (A) S. typhimurium-injected nerves in a mouse allograft schwannoma model showed increased immune cell infiltration (CD45+ common leukocytes and CD68+ pan-macrophages) compared to PBS-injected tumors; yellow arrowheads indicate positive staining (n = 3 mice per group). (B) Quantification of CD45+ cells (left) and CD68+ cells (right) demonstrated significant elevations of leukocytes and macrophages in tumors from VNP20009- and ΔppGpp-injected mice compared to PBS controls. (C) Flow cytometry analysis of tumor-associated macrophages identified as the CD45+F4 / 80+ subset. CD86 expression was used to identify M1 macrophages, while CD206 expression was used to identify M2 macrophages. The M1 to M2 ratio (M1 / M2) was calculated as the % M1 (CD68+) population in CD45+F4 / 80+ divided by the % M2 (CD206+) population in CD45+F4 / 80+. M1 / M2 ratios of TAMs on day 3 (left) and day 7 (right). (D) Adaptive immune cell infiltration of injected tumors was analyzed by flow cytometry 7 days after bacterial injection. The indicated percentages represent CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and CD25+ T cells (CD4+CD25+). Flow cytometry profiles are shown in Figure 10. Quantitative RT-PCR (E) and ELISA (F) of cytokines and inflammasomes in the tumor microenvironment 3 days after S. typhimurium injection. One-way ANOVA was used to compare different treatments. Data are shown as mean ± SEM. N=3 / group. Asterisks (*) indicate differences compared to PBS control; hash marks (#) indicate differences compared to ΔppGpp. * / #p<0.05, ** / ##p<0.01. ** * / ###p<0.001. [Figure 3]VNP20009 injection inhibits tumor growth of primary treated and uninjected distal schwannomas, and the addition of systemic anti-PD-1 mAb enhances primary tumor killing. A) Experimental protocol for combination therapy of VNP20009 and anti-PD-1 mAb. B) 08031-9 cells were subcutaneously implanted into both flanks of FVB / n mice (n=6 mice / group). Significant tumor regression was observed in tumors injected with anti-PD-1 mAb or VNP20009 monotherapy compared with PBS. Combination therapy resulted in significantly greater tumor regression than either VNP20009 or anti-PD-1 mAb monotherapy. C) Injected tumor sites were harvested at the end of the study and analyzed by flow cytometry for cytotoxic CD8+, helper CD4+, and regulatory CD25+ T cells (N=3 / group). D) Anti-PD-1 mAb significantly reduced tumor growth at non-injected sites compared to PBS, while VNP20009 monotherapy and the VNP20009 / anti-PD-1 mAb combination significantly enhanced tumor growth control at non-injected sites compared to anti-PD-1 mAb or PBS-treated groups. E) Non-injected tumor sites were harvested 26 days post-implantation and analyzed by flow cytometry for cytotoxic CD8+, helper CD4+, and regulatory CD25+ T cells (N=3 / group). Flow cytometry profiles are shown in Figure 9. Repeated measures ANOVA was used to compare tumor volume and / or signal between different groups. One-way ANOVA was used to compare flow cytometry data between different groups. Data are presented as mean ± SEM. *p<0.05, **p<0.01. [Figure 4]Injection of VNP20009 inhibited tumor growth of transplanted experimental schwannoma tumors, and the addition of systemic anti-PD-1 mAb enhanced tumor killing. A) Experimental protocol for combination therapy of VNP20009 and anti-PD-1 mAb. B) 08031-9 cells were subcutaneously implanted into the left flank of FVB / n mice (n=6 / group). Significant tumor regression was observed in tumors injected with anti-PD-1 mAb or VNP20009 monotherapy compared with PBS. Combination therapy resulted in significantly greater tumor regression than either VNP20009 or anti-PD-1 mAb monotherapy. C) Injected tumor sites were harvested at the end of the study and analyzed by flow cytometry for cytotoxic CD8+, helper CD4+, and regulatory CD25+ T cells. D) Firefly luciferase-expressing 08031-9FC tumor cells were implanted into the distal sciatic nerve of primary-injected mice 20 days post-implantation (n=5 / group), and tumors were monitored by bioluminescence. E) Secondary tumor sites were harvested 16 days post-implantation and analyzed by flow cytometry for cytotoxic CD8+, helper CD4+, and regulatory CD25+ T cells. Flow cytometry profiles are shown in Figure 10. Repeated measures ANOVA was utilized for the analysis of tumor volume and bioluminescence signal. One-way ANOVA was used for the analysis of flow cytometry data. Data are presented as mean ± SEM. *p<0.05, **p<0.01. [Figure 5] Intratumoral S. typhimurium injection inhibits angiogenesis in allografted mouse schwannoma tumors within the sciatic nerve. VNP20009- and ΔppGpp-injected tumors harvested 2 weeks after bacterial injection showed reduced angiogenesis compared to PBS controls, as determined by both CD31+ staining and direct visualization. Immunohistochemistry was based on three tumors per group; representative staining is shown, with red arrowheads indicating positive endothelial cells. CD31+ cell profiles were quantified using Image-J, and one-way ANOVA was used for data analysis. Data are presented as mean ± SEM. ***p<0.001. [Figure 6]It injection of S. typhimurium suppresses the growth of xenografted human NF1, human sporadic MPNST, and human meningioma subcutaneous tumors. Intratumoral injection of either VNP20009 or ΔppGpp resulted in regression of NF-1-associated (S462TY, A) and growth control of sporadic (STS26T, B) malignant peripheral nerve sheath tumors (MPNSTs) compared with PBS-injected tumors. Similarly, it injection of VNP20009 or ΔppGpp resulted in regression of benign Ben-Men-1 meningioma (C) and growth control of malignant meningioma CH-157 (D) tumor growth compared with PBS-injected tumors. Arrows indicate the time of bacteria / PBS injection. Tumor size between groups was compared using repeated-measures ANOVA. N = 5 mice per group. Data are shown as mean ± SEM. ***p<0.001 comparing PBS with either Salmonella strain. [Figure 7] Intratumoral injection of attenuated S. typhimurium (VNP20009) controls schwannoma development in the human HEI-193 xenograft model and the murine 08031-9 allograft schwannoma model. A) VNP20009 induced significant tumor regression in the xenograft schwannoma model after intratumoral (it) injection 2 weeks after tumor cell implantation (n=8 mice / group). B) VNP20009 injection into the allograft schwannoma model 1 week after tumor cell implantation resulted in growth control (n=8 mice / group). Tumor signals between groups were compared using repeated measures ANOVA. Data are shown as mean ± SEM. *p<0.05, **p<0.01. ***p<0.001. [Figure 8] Schwannoma-bearing nerves injected with Salmonella typhimurium showed increased immune cell infiltration. S. typhimurium-injected nerves in a human xenograft model showed increased immune cell infiltration (CD45+ common leukocytes and CD68+ pan-macrophages) compared to PBS-injected tumors, with positive staining indicated by yellow arrows (n=3 mice / group). [Figure 9]Flow cytometry analysis of splenic macrophages in mice injected with S. typhimurium or PBS. Bacterial-injected immunocompetent schwannoma mice showed an increase in the macrophage population in the spleens of injected mice on day 3 post-injection. Flow cytometry analysis of CD45+F4 / 80+ macrophages showed an increase in the spleens of mice injected with VNP20009 (38.8%) and ΔppGpp (32.4%) compared to PBS (15.4%). [Figure 10] Flow cytometry analysis of tumor-infiltrating immune cells in tumors from mice injected with S. typhimurium or PBS. Analysis of M-1 macrophages (F4 / 80+CD86+) and M-2 macrophages (F4 / 80+CD206+) in tumors harvested on days 3 (A) or 7 (B) after bacterial or PBS injection. (C) Analysis of CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and CD25+ T cells (CD4+CD25+) in treated mice was performed on day 7 after injection of either bacterial strain or PBS. [Figure 11] Flow cytometry profiles of tumor-infiltrating immune cells in tumors from mice injected with VNP20009, PD-1 mAb, VNP20009 / PD-1 mAb, or PBS. Analysis of T lymphocyte infiltration into tumors at the injected (A) and non-injected (B) sites. Tumors were harvested, and staining for CD4+ T cells (CD3+CD4+), CD8+ T cells (CD3+CD8+), and CD25+ T cells (CD4+CD25+) was performed 26 days after injection. [Figure 12] Flow cytometry profiles of tumor-infiltrating immune cells in tumors from mice injected with VNP20009, PD-1 mAb, VNP20009 / PD-1 mAb, or PBS. Analysis of T lymphocyte infiltration into tumors at the injection site (A) and secondary tumor site (B). Tumors were harvested, and CD4+ T cell (CD3+CD4+), CD8+ T cell (CD3+CD8+), and CD25+ T cell (CD4+CD25+) staining was performed 20 days after implantation for primary tumor sites and 16 days after implantation for secondary tumor sites. [Figure 13] Invasion assay of attenuated S. typhimurium in cultured macrophages and schwannoma cell lines. (A) Representative photograph of HEI-193-internalized Salmonella strains after streaking on agar plates for 16 h. (B) Quantification of invasiveness as a percentage of infectivity showed that VNP20009 was more invasive than ΔppGpp but less invasive than wild-type S. typhimurium. The resulting invasion efficiencies of wild-type S. typhimurium in mouse macrophages, human HEI-193, and mouse 08031-9 were approximately 100%, 96%, and 58%, respectively. The invasion efficiencies of VNP20009 and ΔppGpp were significantly lower compared to the wild-type bacteria. The infectivity percentages of VNP20009 in mouse macrophages, human HEI-193, and mouse 08031-9 were approximately 38%, 23%, and 15%, respectively, whereas ΔppGpp showed less invasiveness, with infectivity percentages of 18% for mouse macrophages, 10% for HEI-193, and 5% for 08031-9 cells. [Figure 14] Cytokine ELISA after exposure of cultured human schwannoma (HEI-193, A) or mouse schwannoma (08031-9, B) cell lines to S. typhimurium. Quantification of IL-1β, IL-18, and TNF-α by ELISA after incubation with VNP20009 or ΔppGpp did not reveal any differences in released cytokines compared to PBS-treated cells. One-way ANOVA was used for comparisons between groups. N = 3 independent experiments. Data are presented as mean ± SEM. [Figure 15]Cytokine ELISA after exposure of cultured human (THP-1 differentiated macrophage, A) or murine (RAW264.7 macrophage, B) cell lines to S. typhimurium. Quantification of IL-1β, IL-18, and TNF-α by ELISA after incubation with VNP20009 or ΔppGpp showed significant differences in released cytokines compared to PBS-treated cells. One-way ANOVA was used for comparisons between groups. Data are shown as mean ± SEM. N = 3 independent experiments; *p < 0.05, **p < 0.01. [Figure 16] S. typhimurium did not induce cytokine release in the serum of mice injected directly into tumors in an allograft immunocompetent schwannoma mouse model. ELISA of cytokines in the serum of injected mice was measured 3 days after bacteria administration. One-way ANOVA was used for comparison between groups. Data are presented as mean ± SEM. N = 3 independent experiments. DETAILED DESCRIPTION OF THE INVENTION

[0022] Bacterial-mediated cancer therapy (BCT) using gram-negative organisms was first developed in the mid-19th century by Willi Ian Coley used live Streptococcus pyogenes to treat solid tumors Introduced during treatment 12 Theoretical basis of bacterial cancer therapy The evidence is that the Gram-negative bacterium Salmonella typhimurium (S. S. typhimurium 13~21 Several bacterial strains, including They specifically inhabit hypoxic regions of tumors, proliferate within these regions, and induce direct lysis of tumor cells and anti-tumor immunity. Induce both the establishment of an immune response 22 Furthermore, bacterial injection into tumors is an anti-angiogenic It has been shown to be 23,24 Therefore, bacteria may directly induce cancer cell death. In addition to targeting highly vascularized tumors, it also prevents new tumors from developing. It can act as an immuno-oncological and anti-angiogenic agent that establishes immune control.

[0023] There is a body of preclinical and clinical data supporting BCT as an immunotherapeutic strategy. 20、25 、32、57、58 For 40 years, Mycobacterium bovis Intravesical application of a live attenuated strain of HIV is the only FDA-approved treatment for bladder cancer in situ. there were 29 BCT using an attenuated strain of S. typhimurium is Demonstrated clear efficacy in several preclinical cancer models 16、18~20 . Stillness Attenuated S. typhimurium using intravenous, direct intratumoral, or oral delivery Early-stage clinical trials of )-based BCT have demonstrated safety but have not demonstrated efficacy. Was 25、26、27、28 This lack of effectiveness is due to the rapid division and static nature of cancer cells. This may be due to the use of intravenous delivery. Bacterial inoculation is limited by toxicity with systemic delivery. The lack of efficacy in these studies may be dose-related. There is one BCT approved by the FDA: Mycobacterium bovis (Mycobacterium difficile), which was the standard treatment for non-muscle invasive bladder cancer, A live attenuated strain of B. bovis 29 .

[0024] However, it is possible that BCT may be beneficial for benign neoplasia, possibly because benign tumors tend to be immunologically cold. It has never been suggested as a possibility for 66、67 Therefore, bacterial therapy is the primary Growths such as schwannoma, for which traditional cancer therapies targeting highly replicating cells are ineffective The present invention has not been tested in relation to indolent benign tumors. , a preclinical study supporting bacteriotherapy of schwannoma, a benign neoplasm of the peripheral nervous system. Intratumoral injection of chemokine S. typhimurium directly killed schwannoma cells. It may inhibit angiogenesis and convert the immunological tumor microenvironment from a relatively "cold" to a "hot" one. Furthermore, immunological cell death (if it occurs), immunogenicity, Generation of a pro-immunogenic tumor environment and the combination of VEGF / anti-angiogenesis It was hypothesized that they may synergize to generate an anti-tumor adaptive immune response.

[0025] To test these hypotheses, two attenuated S. typhimurium strains were used. The effects of the strains (VNP20009 and ΔppGpp) were investigated by xenografting in nude mice. Allograft mouse schwannoma tumor models in human NF2 models and syngeneic immunocompetent FVB / N mice The data were evaluated in both attenuated S. typhimurium and showed that intratumoral injection of schwannoma suppressed schwannoma growth in both models. Injection of S. typhimurium into tumor tissue resulted in tumor cell death. This results in the induction of a systemic anti-tumor adaptive immune response in immunocompetent mice. The tumor immune response controlled the growth of non-bacterial-injected tumors that were present at the time of bacterial treatment and demonstrated a "relapse" after treatment. The S. typhimurium challenge prevented the development of tumors. Injected and contralateral non-injected and rechallenged allografts (excluding effects on CD4+ cells) Increases tumor-infiltrating CD4+ helper and CD8+ cytotoxic T cells in schwannoma CD25+ Tregs were reduced, further supporting the existence of an anti-tumor adaptive immune response. Systemic PD-1 immune checkpoint blockade against S. typhimurium Addition of rhizometrium to bacterial injection enhanced schwannoma control in both bacterial-injected and contralateral uninjected tumors. However, rechallenge tumor control of schwannoma was not enhanced. In a study, the effects of attenuated S. typhimurium on schwannomas were investigated. Increased numbers of CD4+ helper T cells and CD8+ cytotoxic T cells and CD25+ A decrease in the number of regulatory T cells was demonstrated.

[0026] In this study, we investigated the effects of two attenuated S. typhimurium strains on the growth of schwannoma. The ability of two strains of B. spp., VNP20009 and ΔppGpp, to attenuate virulence was tested. Thus, both strains have reduced pathogenic potential, including septic shock. The evaluation demonstrated that VNP20009 inhibits the proliferation and proliferation of schwannoma cells in both cultured macrophages and schwannoma cell lines. It is more invasive than ΔppGpp but less invasive than wild-type S. typhimurium. The cultured macrophages were also found to be less invasive than the control macrophages (Fig. 13A and B). Exposure to S. typhimurium strains releases proinflammatory cytokines. However, when VNP20009 and ΔppGpp were co-cultured with schwannoma cell lines, both sites Neither did it induce kine release (Figs. 14A and B and 15A and B). , bacteria-macrophage interactions were observed in S. typhimurium These results suggest that IL-1 may play an important role in the antitumor effects of IL-1.

[0027] In vivo data demonstrate intratumoral efficacy of either VNP20009 or ΔppGpp monotherapy. (it) injection resulted in regression of tumor growth in a human NF2 xenograft model and Furthermore, the magnitude of regression did not differ between the test strains. In a transplanted schwannoma model, i.tVNP20009 injection suppressed tumor growth. The therapeutic effect of VNP20009 was significantly enhanced in the tumor microenvironment compared with ΔppGpp or PBS. Increased apoptotic bodies (Fig. 1D) and IL-18, TNF-α, and IFN-γ This was reflected by increased release of proinflammatory cytokines such as S. thuringiensis (Figure 2E and F). In mice injected with S. typhimurium, elevated systemic cytokine levels were observed. No increase was observed (Figure 16). Further studies showed that the bacterial it in the tumor microenvironment of an immunocompetent schwannoma model after injection of PBS or PBS. We focused on analyzing changes in immune profiles following treatment with HIV.

[0028] M2 macrophages and myeloid-derived suppressor cells (MDSCs) are involved in vestibular schwannoma have been shown to invade the thyroid gland and are associated with aggressive tumor growth 42、59 Tumor-promoting Unlike M2 macrophages, which promote tumor growth, M1 macrophages are immunostimulatory and promote tumor growth. Inhibits tumor growth and shapes adaptive immune responses, at least in part, through phagocytosis and antigen presentation do 60~65 In an allograft schwannoma model, itVNP20 suppresses tumor growth. 009 injection induced CD45+F4 / 80+ tumor-activated macrophages by day 3 after bacterial injection. Among phages (TAM), the ratio of M1 to M2 macrophages increases did.

[0029] Treatment method As shown herein, it has been safely administered to patients with metastatic melanoma and renal cell carcinoma. Attenuated Salmonella strains, such as S. typhimurium, murium) 25、30、31 was effective in treating a mouse schwannoma model. Schwannomas are genetically stable, slow-growing, and highly vascularized with large hypoxic areas. These characteristics make schwannomas an ideal homing environment for bacteria, allowing bacterial cells to grow. It may potentially be a perfect target for cytotoxic and anti-angiogenic properties. The ability of the bacteria to induce an immune response allows for the treatment of multiple distant lesions and the development of tumor-specific antibodies. Typically characterized by a control mechanism that prevents the development of new schwannomas over the patient's lifetime This makes it possible to establish a system.

[0030] The methods described herein include methods for the treatment of benign nervous system tumors. In morphology, the tumor is a schwannoma. Schwannoma tumors are composed of Schwann lineage cells and are distributed to peripheral nerves. These tumors form along the peripheral nerves, spinal cord, and cranial nerves. Compression of the spinal cord and / or brainstem can cause pain, sensory / motor dysfunction, and death. Multiple schwannomas in peripheral distal and intracranial nerves are classified into three types: Transmembrane tumors, neurofibromatosis 1 and 2 (NF1 and NF2), and schwannoma Schwannoma is a benign tumor composed of neoplastic dedifferentiated Schwann cells. Although typically non-malignant and slow-growing, these tumors can have devastating consequences for patients. They can cause extreme pain and affect the senses, including hearing and vision. Schwannomas in NF2 often cause paresthesia, associated with neurological deficits such as weakness or hearing loss, Such tumors often cause excruciating pain. Some schwannomas can grow very large, It can cause compression of adjacent organs or structures, leading to paralysis or progressive compression of the spinal cord or brainstem. Schwannomas are common in NF1, NF2, and schwannomatosis. It may occur sporadically without any genetic predisposition. The incidence of vestibular schwannomas is significant because they are sporadic. , occurring as a single tumor rather than multiple tumors throughout the body. In some embodiments, the subject in need of schwannoma treatment is a patient with neurofibromatosis 1 (NF1); Neurofibromatosis 2 (NF2); Schwannomatosis; Meningioma; Nerve sheath tumor; Schwannoma; Vestibular Schwannoma; sporadic schwannoma; neurofibrosarcoma; neurofibroma; neurofibromatosis (NF); malignant terminal peripheral nerve sheath tumor; and combinations thereof. Subjects who can be treated using the methods of the present invention include subjects who have been diagnosed with These include mammals, e.g., humans, and non-human animal subjects, e.g., cats, dogs, horses, and cats. Examples include gi and ushi.

[0031] The current standard of care for patients with NF2 and schwannomatosis is based on tumor size. The most common treatment is surgical removal or radiosurgery of symptomatic tumors to reduce their size. Surgery is generally effective as long as only tumor is present and the lesion is accessible for resection Unlike sporadic schwannomas, which are treated with multiple tumors, schwannomatous tumors are treated with In NF1 and NF2, resection is difficult due to the inaccessibility of many tumors and the associated major motor impairments. Symptoms include: dizziness, severe sensory loss (including hearing loss in cases of NF2 vestibular schwannoma), and neuropathic pain. This is complicated by both the risk of injury and the risk of trauma. schwannomas in both NF2 and schwannomatosis, and the associated current treatments This painful and debilitating condition, coupled with a lack of treatment options, can lead to substantial morbidity. Therefore, treatment of schwannomas represents a major unmet medical need.

[0032] Generally, the method involves administering a therapeutically effective amount of attenuated Salmonella, such as those described herein. The S. typhimurium described herein is optionally treated with a checkpoint inhibitor. and administering the same in combination to subjects who are in need of, or are deemed to be in need of, such treatment. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, and intratumoral (i In a preferred embodiment, the it route is used to administer the bacteria. Those skilled in the art will recognize that the use of benign nervous system tumors can be beneficial in treating these conditions by maximizing the amount of treatment administered and minimizing potential dose-limiting toxicities (DLTs). In some embodiments, the subject may be identified as having a tumor. The subjects were 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 is a subject who has or has been diagnosed with malignant In some embodiments, the patient does not have a primary solid tumor, e.g., does not have cancer. Elephants may have conditions 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 a dose that effectively treats at least one or more of a disease or disorder. refers to the amount of a composition required to relieve multiple symptoms, sufficient to provide the desired effect. Thus, the term "therapeutically effective amount" refers to a therapeutically effective amount of a pharmaceutical composition. This refers to an amount of a composition sufficient to provide a specific anti-tumor effect when administered to an animal. As used herein, an effective amount refers to an amount that, in various circumstances, delays the onset of symptoms of a disease, or prevents the onset of symptoms of a disease. Altering the course of symptoms (for example, but not limited to, slowing the progression of disease symptoms) or an amount sufficient to reverse the symptoms of the disease. However, for any given case, it is generally not feasible to specify Such an "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation. By administering a therapeutically effective amount of the compounds described herein for the treatment of tumors, e.g. tumor size, tumor number, tumor growth rate, or likelihood of recurrence after treatment with the methods described herein. This can result in a decrease in

[0034] Therefore, the methods of the present invention provide for the use of attenuated S. typhimurium ( As shown herein, the administration of a S. typhimurium strain enhances the efficacy of the treatment. Therefore, in a preferred embodiment, the method involves intratumoral bacterial concentration rather than intravenous delivery. Intratumoral injection of bacteria can be utilized to increase efficacy and minimize systemic toxicity. Direct injection of attenuated S. typhimurium into schwannomas, as shown in This resulted in a vaccine-like effect and induced an anti-tumor adaptive immune response.

[0035] Attenuated S. typhimurium As used herein, the term "attenuated" refers to a bacterial strain that is attenuated compared to the native strain. It has been made less virulent and therefore harmless or less virulent. Attenuation does not mean inactivation. Attenuation means that both strains The current data are primarily based on VN Although P20009 and ΔppGpp are described, other attenuated strains can also be used. Methods for generating attenuated Salmonella strains include, for example, directed or random bombardment. Methods known in the art, such as spontaneous mutagenesis followed by screening for reduced virulence, can be used. For example, the aroA gene (aroA connects glycolysis to the synthesis of aromatic amino acids) in Shikimi aroA-deficient Salmonella strains are described, for example, by Feigner et al. al, mBio, 2016, 7: e01220-16); the gene purl (defective in purine synthesis) or asd gene (aspartic acid semialdehyde dehydrogenase required for cell wall synthesis) Directed mutations of Salmonella (defective in hydrogenase) can be used. Attenuated strains of Salmonella are disclosed in International Publication No. WO 2014 / 005683; International Publication No. WO 2014 / 005683; Publication No. 2016 / 202459 Brochure; International Publication No. 2013 / 09189 Pan FRET; and U.S. Patent Application Publication No. 20200038496 (attenuated S. typhi). The strains that can be used in the current method are S. typhi Ty21a. , Salmonella enterica serovar typhimurium urium) ("S. typhimurium"), Salmonella Montevideo (S Salmonella montevideo, Salmonella enterica serovar typhi serovar Typhi ("S. typhi"), Salmonella enterica serovar para Salmonella enterica serovar Paratyphi B ("S. paratyphi B" typhi B), Salmonella enterica serovar paratyphi C (Salmonella enterica ser Paratyphi C), Salmonella enterica Salmonella enterica serovar Hadar ("S. hadar") "), Salmonella enterica serovar Enteritidis (Salmonella enterica serova Enteriditis) ("S. enteriditis"), Salmonella enterica Salmonella enterica serovar Kentucky ("S. Kentucky") S. kentucky), Salmonella enterica serovar infantis S. enterica serovar Infantis ("S. infantis"), monkey Salmonella enterica serovar Pullorum ("S Salmonella enterica serovar gallinarum (S. pullorum) enterica serovar Gallinarum ("S. gallinarum"), Salmonella Salmonella enterica serovar Muenchen ("S. S. muenchen), Salmonella enterica serovar anatum terica serovar Anaturn) ("S. anatum"), Salmonella enterica Salmonella enterica serovar Dublin ("S. dublin") n)), Salmonella enterica serovar Derby ) ("S. derby"), Salmonella enterica serovar cholera Suis Kunzendorf (Salmonella enterica serovar Choleraesuis var. Kunzendorf) (" S. cholerae kunzendorf), and Salmonella Salmonella enterica serovar minnesota ("S. Minnesota") (S. minnesota)). For example, attenuated versions of S. No. 2008 / 039408 and U.S. Patent Application Publication No. 2020002305 3; U.S. Patent Application Publication No. 20190153452; U.S. Patent Application Publication No. No. 20170333490 and U.S. Patent Application Publication No. 20180339032 Specifications;Grant et al., PLoS Pathog. 2012 Dec; 8(12): e1003070;Tennant and Levine, V accine. 2015 Jun 19; 33(0 3): C36-C41.

[0036] In a preferred embodiment, the attenuated strain used in the present method is Clostridium novyi. (Clostridium novyi) (see, for example, International Publication No. 2014160950, brochure) In a preferred embodiment, the attenuated strain used in the present method is an intracellularly induced A lysis gene or cassette operably linked to a Salmonella promoter (See, e.g., U.S. Patent Application No. 20170333490).

[0037] Combination therapy The methods of the present invention involve the use of attenuated Salmonella in combination with one or more other treatments. For example, current studies have demonstrated that the administration of HIV-1 strains of the bacterium Epstein-Barr virus (EBV) in immunocompetent mice Tumor helper CD4+ and cytotoxicity by itVNP20009 in schwannoma Increased percentage of CD8+ T cells and concomitant increase in percentage of CD25+ Tregs This resulted in a shift to M1 tumoricidal macrophages. These changes in tumor-infiltrating T cell populations associated with S. typhimurium ) suggests an anti-tumor adaptive immune response induced by schwannoma. -L1 expression indicates resistance to cell-mediated immunity in the tumor-mediated immune microenvironment 49 This Taking this into account, the effects of PD-1 immune checkpoint blockade on itS S. typhimurium (VNP20009)-associated schwannoma growth regulation and host The effect of this combination on the development of anti-tumor adaptive immunity was evaluated. The data showed that this combination CD4+ helper and CD8+ cytotoxic T cells infiltrating both injected and uninjected tumors Increased numbers of CD25+ regulatory T cells associated with decreased numbers of bacterially injected schwannomas The results showed that the tumor regression was enhanced compared with that of the bacterial-injected tumors (Figs. 3 and 4). In the contralateral non-bacterial injected schwannoma (Figure 3D, E) and rechallenge schwannoma (Figure 4D, E), Combining VNP20009 with anti-PD-1 mAb showed the same enhancing effect on T cell populations Results were obtained (increase in CD4+ and CD8+, decrease in CD25+), but VNP2000 There was no difference in proliferation inhibition between anti-PD-1 mAb and VNP20009 alone. , itVNP20009 and VNP20009 / anti-PD-1 for schwannoma growth control The efficacy of both mAb combinations was significantly greater in rechallenged than in primary bacterial-injected tumors (Figure 4B). The tumors (Figure 4D) appear to be larger. Differences in the biology of non-injected contralateral schwannomas and rechallenged schwannomas remain to be elucidated. Not yet.

[0038] Thus, the methods of the present invention include the use of checkpoint inhibitors, such as PD-1 signaling inhibitors. inhibitors of these, e.g., antibodies that bind to PD-1, CD40, or PD-L1; or Inhibitors of Tim3 or Lag3, e.g., antibodies that bind to Tim3 or Lag3 or administering a combination of an antibody that binds to CTLA-4 and bacteria (together or separately). This may include:

[0039] Exemplary anti-PD-1 antibodies that can be used in the methods described herein include human PD-1 antibodies. PD-1; an exemplary PD-1 protein sequence is Exemplary antibodies include those provided under accession number NP_005009.2. , U.S. Patent No. 8,008,449; U.S. Patent No. 9,073,994; and U.S. It is described in the specification of Patent Application Publication No. 20110271358, and is designated as PF-068015. 91, AMP-224, BGB-A317, BI754091, JS001, MEDI0 680, PDR001, REGN2810, SHR-1210, TSR-042, Pembu These include lorizumab, nivolumab, avelumab, pidilizumab, and atezolizumab. .

[0040] Exemplary anti-CD40 antibodies that can be used in the methods described herein include human CD40 antibodies. an exemplary CD40 precursor protein sequence is NCBI accession numbers NP_001241.1, NP_690593.1, NP_ 001309351.1, NP_001309350.1 and NP_00128968 2.1. Exemplary antibodies include those described in WO 2002 / 088186. Brochure; International Publication No. 2007 / 124299 Brochure; International Publication No. 2011 / 123489 Brochure; International Publication No. 2012 / 149356 Brochure; Country International Publication No. 2012 / 111762; International Publication No. 2014 / 070934 Specification; U.S. Patent Application Publication No. 20130011405 Specification; U.S. Patent Application Publication No. 20 No. 070148163; U.S. Patent Application Publication No. 20040120948; U.S. Patent Application Publication No. 20030165499; and U.S. Pat. No. 8,591,900 These include those described in the specification, such as dacetuzumab, lucatumumab, bleselumab, Teneliximab, ADC-1013, CP-870,893, Chi Lob 7 / 4, HCD122, SGN-4, SEA-CD40, BMS-986004, and APX0 In some embodiments, the anti-CD40 antibody is a CD40 agonist. It is not a CD40 antagonist.

[0041] Exemplary anti-CTLA-4 antibodies that can be used in the methods described herein include , including those that bind to human CTLA-4; exemplary CTLA-4 protein sequences. The columns are provided under NCBI accession number NP_005205.2. For antibodies, see Tarhini and Iqbal, Onco Targets Ther. 3:15-25 (2010); Storz, MAbs. 2016 Jan; 8(1): 10-26; U.S. Patent Application Publication No. 2009025274; U.S. Patent No. 7605238; U.S. Pat. No. 6984720; European Patent No. 12124 No. 22; U.S. Pat. No. 5,811,097; U.S. Pat. No. 5,855,887 ;U.S. Provisional Patent Application No. 6,051,227; U.S. Patent No. 6,682,736; European No. 1,141,028; and U.S. Pat. No. 7,741,345; These include ipilimumab, tremelimumab, and EPR1476. do.

[0042] Exemplary anti-PD-L1 antibodies that can be used in the methods described herein include: PD-L1; an exemplary PD-L1 protein sequence is: NCBI accession numbers NP_001254635.1, NP_001300958 .1, and NP_054862.1. Exemplary antibodies include those disclosed in U.S. Pat. Patent Application Publication No. 20170058033; International Publication No. 2016 / 061142 Brochure; International Publication No. 2016 / 007235 Brochure; International Publication No. 2014 / No. 195852; and WO 2013 / 079174 It is listed in BMS-936559(MDX-1105), FAZ053, KN0 35, atezolizumab (Tecentriq, MPDL3280A), avelumab (Ba vencio), and durvalumab (Imfinzi, MEDI-4736). can be.

[0043] Exemplary anti-Tim3 (Hepatitis A virus) antibodies that can be used in the methods described herein are: The antibody (also known as virus cell receptor 2 or HAVCR2) binds to human Tim3. An exemplary Tim3 sequence is available under NCBI accession number NP Exemplary antibodies include those provided in WO 201607 1448; U.S. Pat. No. 8,552,156; and U.S. Patent Application Publication No. No. 20180298097; U.S. Patent Application Publication No. 20180251549 Publication No. 20180230431; Publication No. 2018 0072804; U.S. Patent Application Publication No. 20180016336; U.S. Patent U.S. Patent Application Publication No. 20170313783; U.S. Patent Application Publication No. 201701141 35; U.S. Patent Application Publication No. 20160257758; U.S. Patent Application Publication No. US Patent Application Publication No. 20160257749; US Patent Application Publication No. 20150086574 and U.S. Patent Application Publication No. 20130022623, These include 3321367, DCB-8, MBG453 and TSR-022.

[0044] Exemplary anti-Lag3 antibodies that can be used in the methods described herein include human Exemplary Lag3 sequences are available from NCBI accession no. Exemplary antibodies include those listed 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 No. 20180326054; U.S. Patent U.S. Patent Application Publication No. 20180251767; U.S. Patent Application Publication No. 201802304 31; U.S. Patent Application Publication No. 20170334995; U.S. Patent Application Publication No. 201 70290914; U.S. Patent Application Publication No. 20170101472; U.S. Patent Application Publication No. 20170022273; U.S. Patent Application Publication No. 20160303 124 and includes BMS-986016.

[0045] The methods of the present invention also include administering a combination of an angiogenesis inhibitor and a bacterium (together or separately). Vascular endothelial growth factor (VEGF), its receptor (VEGFR), and Many angiogenesis inhibitors are known, including those that target other molecules involved in angiogenesis. Specific examples include axitinib (INLYTA); bevacizumab (AVAS); TIN); cabozantinib (COMETRIQ); everolimus (AFINITOR); Lenalidomide (REVLIMID); lenvatinib mesylate (LENVIMA); Panib (VOTRIENT); ramucirumab (CYRAMZA); regorafenib (ST IVARGA); sorafenib (NEXAVAR); sunitinib (SUTENT); domide (SYNOVIR, THALOMID); vandetanib (CAPRELSA); or aflibercept (Ziv-aflibercept) (ZALTRAP). 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; See Goel et al., Cold Spring Harb Perspect Med. 2012 Mar;2(3):a006486.

[0046] Alternatively or additionally, the methods 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 typhimurium described herein Salmonella strains are administered to patients undergoing surgical removal or partial removal of neoplasms or tumors, such as schwannomas. The various treatment methods of the present invention can be administered before, simultaneously with, or after the selective removal of the steroid hormone. Treating the subject with chemotherapy, surgery, radiation therapy, or chemotherapy, or a combination thereof. The method may further include:

[0047] Pharmaceutical Compositions and Methods of Administration The methods described herein include the preparation of pharmaceuticals containing attenuated Salmonella as an active ingredient. The present invention includes the use of a pharmaceutical composition. A pharmaceutical composition typically includes a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" means a carrier that is compatible with pharmaceutical administration. These include saline solutions, solvents, dispersion media, and the like. Supplementary active compounds also include those described, for example, in the art. and / or as discussed herein, e.g., Che can be incorporated into compositions such as checkpoint inhibitors and / or angiogenesis inhibitors .

[0048] A pharmaceutical composition is 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 include: [Example]

[0049] The invention is further described in the following examples, which are provided to illustrate, but are not to be construed as limiting the scope of the invention. The scope of the invention described is not intended to be limiting.

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

[0051] cell culture HEI-193 human schwannoma cell line (House Ear Institute, Lo (from DJ Lim of Angeles, CA) was established from a schwannoma of an NF2 patient. were immortalized with the human papillomavirus E6 / E7 genes and propagated as described. made 68、69 Mouse 08031-9 schwannoma cells (University of California) Marco Giova of Ornia, Los Angeles, CA (from Dr. nnini) and grown as described 50 The cell lines were then incubated with bioluminescent Fluc (firefly luciferase) and m for imaging and IHC, respectively. infected with a lentivirus encoding Cherry 70 Human MPNST (STS. 26T) cells were provided by Dr. David Largaespada (University of f Courtesy of the Masonic Cancer Center, Minnesota were provided and grown as described 56 Human NF-1-associated MPNST (S4 62TY) cells were kindly provided by Dr. Timothy P. Cripe (Nationwide Chi The data were kindly provided by the Children's Cancer Center at the University of Illinois's Hospital. Ben-Men-1 and CH-157 cells were cultured in the Long-Sheng Ch Dr. Ang (children at Nationwide Children's Hospital) Cancer Center) and Dr. G. Yancey Gillespie (University of Kindly provided by the University of Alabama Birmingham. Phorbol-12-myristate 13-acetate for macrophage differentiation (PMA) (Sigma-Aldrich, USA) to human monocytes at a final concentration of 100 μM After 24 hours, the PMA-supplemented medium was removed, the cells were washed with PBS, and the macrophages were incubated for 24 hours. To obtain phenotypic characteristics of the cells, the cells were left in untreated PMA-free medium for an additional 24 hours. 71 . Murine RAW macrophages were obtained (ATCC, USA). All cell lines were cultured in RPMI medium according to the manufacturer's instructions. It was confirmed that there was no contamination with mycoplasma.

[0052] bacterial culture Attenuated Salmonella enterica serovar Typhimurium strain VNP20009 (modified lipid A (msbB-), with a purine auxotrophic mutation (purI-) was purchased (AT CC, USA, Cat. No. 14028), ΔppGpp strain (deficient in ppGpp synthesis) (RelA::cat, SpoT::kan)) were kindly provided by Karste Dr. Tedin, Institute for Microbiology and Epizootics, Center for Infection Medicine Bacterial cells were grown under aerobic conditions as previously described, with low NaCl content. Luria-Bertani (LB) broth medium containing thorium (Difco Laboratories) The cells were cultured overnight at 37°C and 300 rpm in a 1000-kJ / 2000 rpm oven (Yates, USA). 31、47 To summarize: The cells were grown to late logarithmic phase (OD600nm = 0.8) and incubated at 5000 rpm for 10 min. The cells were collected by centrifugation for 1 min, washed twice with sterile 1x phosphate-buffered saline (PBS), and then Injection into tumors or infection of cultured cells was performed.

[0053] animal All animal studies were performed under the supervision of Massachusetts General Hospital l (MGH, Boston, MA) Institutional Animal Care and Use Committee (IACUC protocol) This study was approved by and conducted under the supervision of the National Institute of Health Sciences (NIHS) (No. 2014N000211). Ages 5-7 weeks Male mice, nu / nu and FVB / N (Charles River Laboratories) atories) with free access to food and water and a 12:12 light / dark cycle. and the Center for Comparative Medicine at MGH. Daily health checks were performed by staff / veterinarians.

[0054] Animal model and intratumoral bacterial injection Sciatic schwannomas were implanted into the left sciatic nerve of isoflurane-anesthetized mice using HE as described. Direct injection of I-193FC human or 08031-8FC mouse schwannoma cells Therefore, 72 HEI-193FC or 08031-9FC cells were treated with trypsin. Wash the cells, rinse with cold PBS, and then add 0.5 μl of 30,000 μL of PBS (or 08031-9 Cells (10,000 for FC) were placed in a glass micropipette and gas-operated micropipette. An injector (IM-300; Narishige, Tokyo, Japan) was used. and athymic nude mice (nu / nu, 5-7 week old male, National C Cancer Institute [NCI]), or syngeneic FVB / N mice (5–7 Injection into the sciatic nerve of a week-old male mouse (Charles River Laboratories) Two weeks after HEI-193 tumor cell implantation or one week after 08031-9 tumor cell implantation The tumor was then injected with 2 μl of PBS [9, 10 of 13 4 CFU attenuated S. typhimurium (VNP200 09 or ΔppGpp). Tumor growth was monitored using HEI-1 as described. In vivo bioluminescence imaging was performed weekly for 93 and twice weekly for 08031-9. monitored by 72 Briefly, mice were intraperitoneally injected with the Fluc substrate d-luciferin. After 10 minutes, the patient was injected intravenously with a highly efficient IVIS spectrum (Caliper Life Sciences). Signals were acquired using a chemiluminescence (CT) imaging system (BioNTech, Hopkinton, MA). In the case of 08031-9, resuspend in DMEM and add Matrigel (BD Biosciences The cells were mixed with the IgG (1:1) and then implanted subcutaneously (sc) into FVB / N syngeneic mice. For NF-1 or meningioma xenograft models, human S462TY, STS.26T, and Be n-Men-1 or CH-157 cells were mixed with Matrigel (1:1) and cultured as described. The cells were subcutaneously transplanted into nu / nu mice. 56 The tumor volume was calculated using the formula (W × L × L × π / 6 ) (where width (W) and length (L) are the two largest diameters) 50 . The tumor is 150 mm 3 Once this was reached, intraperitoneal (ip) PD-1 mAb (250 μg / injection) was administered. Treatment with IV intravenous injection (Bio X Cell, USA) was initiated and repeated every 3 days as described above. returned 51 . VNP20009 injection (10 4 CFU / 100 μl PBS) The injection was administered intratumorally using a phosphorus syringe. Control mice were injected with PBS on the same schedule. Treatment was with vehicle or isotype antibody.

[0055] Histological and immunohistochemical analyses Animals were terminally anesthetized with isoflurane (3%) and sacrificed by decapitation. Tumor tissue was removed and analyzed by hematoxylin and eosin (H&E) and immunohistochemistry. Snap-frozen for histochemical staining 73 The tumors were kept in OCT blocks at -80°C. Sections were stained with H&E according to routine protocols. Proliferation marker staining was performed using Ki 67 (Abcam, Cambridge, MA) was used as an antibody against CD. 45, antibodies against CD68 and CD31 were used to detect leukocytes, macrophages, and All antibodies were used for angiogenesis staining (Abcam, Cambridge, MA). ) was purchased. Briefly, sections were dried overnight at room temperature (RT). Sections were then dried at 4°C for 1 The sections were fixed in pre-cooled acetone for 10 min, dried, and then immediately stained. and blocked with serum-free protein block (Dako, Carpinteria, CA). The sections were blocked and peroxidase was quenched with double endogenous enzyme block (Dako). After washing with PBS, the cells were incubated with the primary antibody for 1 hour at room temperature, and then washed with PBS. Wash and incubate with horseradish peroxidase-conjugated secondary antibody for 3 min at room temperature (RT). The sections were washed with PBS and then incubated with DAB solution (Dako). Counterstaining was performed by immersing the sections in ethanol and xylene, followed by Cytochrome P450 staining. seal(Richard Allan Scientific, San Diego, This is achieved by mounting the cells in a vitreous solution (e.g., vitreous saccharin) and covering them with a coverslip for visualization under a microscope. In vivo apoptosis staining was performed using TACS2 TdT-DAB in situ apoptosis staining. The assay was evaluated using a genotyping kit (Trevigen, Gaithersburg, MD). After mounting on slides, the specimens were soaked in 3.7% formaldehyde according to the manufacturer's instructions. 15 μm sections (cryostat) of fixed, untreated, frozen nerves were stained and visualized under a light microscope. Visualization was performed with aminobenzidine (DAB). The antibodies utilized are provided in Table 1.

[0056] [Table 1]

[0057] Measurement of cytokine RNA in tumors using real-time quantitative (qRT-PCR) Three days after bacterial injection, tumor tissues were excised and RNA was extracted using Trizol. Script(TM)IV VILO(TM) with ezDNase enzyme Total RNA was transcribed into cDNA using a PCR kit (Invitrogen). Incubate at 7°C for 10 minutes to digest the DNA, followed by PCR with the ProFlex PCR system. Stem, Applied Biosystems, USA) at 25°C for 10 minutes, 50 The qPCR reaction was incubated at 85°C for 5 minutes. Use the wells as input and measure the expression of the target gene using TaqMan probes. The qPCR assay was performed using an Mx3000P qPCR system (Agilent Technologies, USA) using standard cycling mode conditions. Melting curve analysis was performed using MxPro qPCR software to identify the number of nucleotides in each plate. Primer efficiency was verified to rule out non-specific amplification. The difference in cycle threshold (Ct) values ​​between the 2 and 3 ΔCt was used to calculate relative expression using the 2-ΔΔCt method. Fold changes were calculated by converting and comparing samples. All reactions were performed in triplicate. .

[0058] Measurement of cytokine proteins in tumors using ELISA On day 3 after bacterial injection, tumor tissues were excised and lysed in NP40 lysis buffer containing proteinase inhibitors. The mixture was homogenized with ethanol and centrifuged at 13,000 rpm for 10 minutes to recover the supernatant. Cytokine levels were analyzed using individual Quantik assays for humans and mice according to the instructions of ine ELISA kit (R&D systems, Minneapolis) was used. Measured using: interferon gamma (IFN-γ) (BD bioscience) , TNF-α (BD bioscience), IL-1β / IL-1F2 (BD bi Bioscience) and IL-18 (BD Bioscience). using a spectrophotometer (SpectraMax, Molecular Devices) Measure the substrate color reaction at 450 nm with the compensation wavelength set to 540 nm or 570 nm. Afterwards, the results were quantified with a standard curve.

[0059] In vitro invasiveness assay Macrophages (human THP-1 and mouse RAW264.7 macrophages) and Schwannoma (human HEI-193 and mouse 08031-9) cells were cultured in 24-well plates. 10 per well in a culture plate 4 The cells were grown to a density of 1000 μg / ml. The cells were washed with warm PBS. In parallel, bacterial cells were cultured at logarithmic growth rates as previously described. Grown to late growth phase and diluted in cell culture medium to a multiplicity of infection of 50:1 bacteria / cell ( The MOI (multiple infections per 1000 cells) was shown. Cultured macrophages and schwannoma cells were added with the medium containing bacteria, and the cells were incubated for 3 days. The cells were then placed in a 7°C incubator for 60 minutes. Wash with PBS and incubate in medium containing gentamicin sulfate (50 μg / mL) for 30 min. The cells were then incubated for 1–2 h to kill any extracellular bacteria attached to the cell surface. After rinsing five times with 2 mL of PBS, add 0.2 mL of 0.1% Triton X-100 to the wells. The cells were lysed and the attached bacteria were detached by adding LB broth (0.8 mL) for 0 min. ) was added and each sample was mixed vigorously to prepare a homogenous suspension for serial dilution. The solution was prepared, plated on LB agar medium, and incubated overnight at 37°C to allow for colony formation. The colony-forming units (CFU) were counted.

[0060] Flow cytometry Tissues were collected from mice (n = 3 / group) and lysed in freshly prepared lysis buffer (20 mM He 125 U / mL collagenase type XI, 60 U / mL hyaluronan in PBS containing pes Ionidase type I, 60 U / mL, DNase 1, and 450 U / L collagenase I (Sigma-Aldrich) in a water bath at 37°C for 1 hour. Cells were dissociated by gently flicking the plate every 10 minutes for crystallization and cell dissociation. The cell suspension was passed through a pre-wetted strainer, a 70 μm cell strainer (BD-Fal The suspension was mixed with 10 uL of trypan blue. After quantifying the cells, the cell suspension was incubated at 2000 rpm for 10 min at 4°C. Remove the lysis buffer by centrifugation at 10 °C, wash and resuspend in 1x PBS. 6 cells / 10 The cells were then incubated with 2 μL of FC blocking agent (BD Bioscience The cells were then washed with PBS and then incubated in PBS for 15 minutes at room temperature. Ink for 1 hour in the dark with fluorescently labeled antibodies against various immunomarkers or immunoglobulins. After incubation with PBS, the cells were then washed with 2% paraformaldehyde (PFA solution). ) overnight. The following antibodies against mouse immune markers were used for surface staining: D45, F4 / 80, CD206, CD86, LY6G, NK1.1, NKp46, CD CD11b, CD11c, CD4, CD8, CD3, CD25. FACS and analysis were performed using FACS. CSDiva software (BD Bioscience) and FlowJo software The analysis was performed using FACSAria and LSRFortessa software. The antibodies utilized are provided in Supplementary Table 1.

[0061] Data analysis All data are expressed as group mean ± standard error of the mean (SEM). Data were analyzed using ad Prism and Microsoft Excel. Repeated measures analysis of variance (ANOVA) was used to assess tumor volume and / or phenotype as described. or compared signals74 One-way ANOVA was used to evaluate cytokine expression and The MRI and flow cytometry data were analyzed. P<0.05 was considered significant.

[0062] [Example 1] Intratumoral attenuated S. typhimurium injection resulted in the development of tumors in xenografted humans and in the control group. Tumor growth is suppressed in a mouse schwannoma seed transplant model Intratumoral (it) injection of S. typhimurium resulted in nu / nu immunization. Human (HEI-193) Developing in the Sciatic Nerve of Deficient and FVB / N Immunocompetent Mice cell line) and mouse (08031-9 cell line) schwannoma growth can be controlled. Two different strains of S. typhimurium were evaluated for their Two strains, namely VNP20009 and ΔppGpp, were evaluated. Both strains were used in preclinical studies. 1 9、32、33 VNP20009 is currently undergoing clinical trials. 25、34 Even higher tumor fingers mutant attenuated forms of wild-type bacteria that exhibited tropism and improved safety profiles .

[0063] Tumor burden was measured using HEI-193FC (human NF2 schwannoma) and 08031-9FC (human NF2 schwannoma). Immunohistochemical analysis of firefly luciferase (Fluc) expressed by mouse NF2-deficient schwannoma cells Once the tumor signal was stable (HEI-1), the tumor was evaluated via in vivo bioluminescence imaging. Approximately 2 weeks or 1 week after tumor implantation of 93FC or 08031-9FC cells, respectively 1A and B), tumor-bearing sciatic nerves were directly visualized and infected with attenuated S. typhimurium ( S. typhimurium) (VNP20009 or ΔppGpp) or PBS (control) Tumor growth was further investigated in nude mice bearing human NF2 schwannoma. Xenografts were performed in NF2-bearing immunocompetent FVB / N mice for 5 weeks and followed for 2 weeks. In the schwannoma model, the study ended 7 weeks after tumor cell implantation—at which point bacterial injection Most of the tumors had no bioluminescent signal. The termination of the study was determined by the onset of motor dysfunction in the tumor-bearing hind limbs of control mice ( The animals were evaluated by both the individual groups and animal care technicians, all of whom were blinded to the study groups. Two additional replicates of this study were performed in xenograft models, one replicate Allograft models were performed (n = 8 mice / group for all studies; Figures 7A and B). ).

[0064] Intratumoral injection of attenuated S. typhimurium VNP20009 strain Thus, all three replicates in the xenograft human NF2 schwannoma model (p<0.01, 1A; 7A) and replication of both in an allograft mouse schwannoma model (p<0.0 5, Figure 1B; Figure 7B), there was a decrease in the bioluminescent tumor signal compared to the PBS control. The growth curves of bacteria- and PBS-injected mice were as early as 10 days after VNP20009 injection. Both tumor models began to branch within one week. Bacterial treatment resulted in increased branching at two weeks after bacterial injection. By the end of the study, HEI-193FC tumor signals had regressed to undetectable levels in 5 of 8 mice. Across the three replicates of this experiment, 75% of VNP20009-injected animals ( 18 / 24 mice) had no detectable tumor signal by the end of the experiment ( Figure 1A, Figure 7A).

[0065] Complete regression of tumor signals after bacterial injection was observed in an allograft mouse schwannoma model. However, tumor growth control continued until the time of sacrifice, at which time V was significantly higher compared to the PBS control. In NP20009-injected mice, the bioluminescence signal was approximately eightfold lower ( Fig. 1B , Figure 7B).

[0066] Next, the ΔppGpp strain of S. typhimurium was identified as VNP20009. In a xenograft model, we investigated whether IL-16 had a similar therapeutic effect to that of IL-16. The effects of VNP20009 and ΔppGpp on the expression of α-glucan were indistinguishable from each other, but were not significant in allografted mice. In a schwannoma model, the ΔppGpp strain of S. typhimurium VNP20009 suppressed tumor growth (p<0.05 vs. VNP20009) BS, Fig. 1B), and indeed, there was a significant difference between the two strains (p<0.05 VNP20 009 vs. ΔppGpp, Fig. 1B ).

[0067] At the end of the experiment (i.e., 5 weeks after bacterial injection in the xenograft human schwannoma model, and allograft model), In the transplanted mouse schwannoma model, tumor-bearing nerves (n=3 mice) were collected 2 weeks after bacterial injection. Histological analysis of VNP20009 and ΔT cells (mouse / group) showed that tumors were significantly more sensitive to VNP20009 and ΔT cells than PBS-injected tumors. Both ppGpp-injected tumors showed abundant apoptotic bodies (Fig. 1C and D). Quantification of tissue from the seed transplant model revealed that VNP20009-injected schwannoma (570 ± 77 ;p<0.0005) and ΔppGpp-injected schwannoma (230±68, p<0.005 ) revealed a higher number of apoptotic bodies than PBS-injected tumors (4 ± 1). Comparing the apoptotic bodies induced by the two bacterial strains, H Compared with ΔppGpp-treated EI-193 schwannoma tumors, VNP20009-treated tumors The number of apoptotic cells was higher in xenografted human mice (p<0.01, Figure 1C). In NF-2 schwannomas, apoptosis was greater after VNP2009 injection than after ΔppGpp injection. In the allograft mouse schwannoma model, consistent differences between groups were observed. observed in ΔppGpp-injected tumors (110±27, p<0.01) and PBS-injected tumors. (6±0.3, p<0.001) compared with VNP20009-injected tumors (340±63 , ) had more apoptotic cells. ΔppGpp-injected tumors were significantly larger than PBS-injected tumors. The number of apoptotic cells was significantly higher than in the control (p<0.01, Figure 1D). In the control group, itVNP20009 injection increased apoptosis by approximately three times that of ΔppGpp injection. This resulted in more ptotic cells (p<0.01, Figure 1D).

[0068] [Example 2] Allograft mice were injected with attenuated S. typhimurium. Increased levels of immunogenic cytokines and altered immune cell infiltration were observed in the schwannoma model. be fooled One of the most exciting properties of BCT is its ability to induce anti-tumor adaptive immunity. 35~37 Based on this finding, we investigated the efficacy of attenuated S. typhimurium in the prevention of HIV infection. It is thought that transcutaneous infection of schwannoma may have a vaccination effect, inducing antitumor adaptive immunity in the host. Immunotherapy for schwannoma is expected to be effective in affected individuals who typically have multiple tumors and continue to receive treatment throughout their lives. New tumors can develop through the spleen and be surgically removed without substantial risk of major nerve damage. The tumor is in a location where it is difficult to remove, complete resection is often not feasible, and This is especially true given that tumors require multiple surgeries to develop throughout a patient's life. worth it.

[0069] It injection of bacteria into xenograft and allograft schwannoma models induces apoptosis Although cell death is induced (Fig. 1), we have not observed pyroptosis and / or immunogenic cell death. We also wanted to investigate whether there was evidence of cell death. and ΔppGpp were grown in the sciatic nerves of nude and immunocompetent mice, respectively. Inject it into human HEI-193 and mouse 08031-9 schwannoma tumors. and may induce a wide range of indices of the host's innate and adaptive immune responses. Common lymphocyte antigen marker CD45 38 and the monocyte and tissue macrophage marker CD68 3 9 Intratumoral immune cell infiltration was assessed through analysis of xenografted human NF-κB. Five weeks after bacterial injection in the two models, and two weeks after bacterial injection in the allograft mouse schwannoma model. Immunocytochemical staining of tumors collected at 1 h (n=3 tumors / treatment / model) was assessed. The timing of sacrifice is determined for the reasons mentioned above, i.e., in most mice (xenograft models), The tumor signal dissipates or precedes severe pathology in control mice (allograft models). It was selected for the following reasons:

[0070] In xenograft models, histological analysis of tumor-bearing nerves revealed that both classes of cells Compared with PBS-injected tumors, which did not show any CD45+ leukocytes and CD68+ macrophages, Phage-rich tumor infiltration was evident (Figure 8). The same analysis of tumors was performed by it injection of either VNP20009 or ΔppGpp. In comparison with PBS-injected tumors, CD45+ leukocyte and CD68+ macrophage infiltration The results showed that C in the tumor microenvironment of the allograft model increased (Figure 2A). Quantification of D45+ and CD68+ cells revealed that this bacterial injection significantly outnumbered PBS-injected controls. showed an increase in tumor-infiltrating leukocytes and macrophages compared with control (Fig. 2B).

[0071] Using only an allograft model in which these tumors develop in immunocompetent host mice, S. We focused on characterizing Schwannoma killing by S. typhimurium. We investigated whether there were indicators of immunogenic cellular responses and immunogenic cell death. M1 tumoricidal and M2 tumorigenic (M1) 40 Host anti-tumor Promotion of adaptive immunity or (M2) 41 Differential inhibition of host anti-tumor adaptive immunity It appears that the key determinants of host antitumor immunity are the differentiation of M1 and M2 macrophages. Human schwannomas are composed of up to 50% macrophages depending on the cell number. It is reported to be configured 42 The higher the macrophage content, the higher the tumor growth rate. becomes higher 43 Three days after it injection of attenuated S. typhimurium and 7 days later, CD45+F4 / 80+ collected from intrasciatic nerve allografted mouse schwannoma. CD86+ (M1 type, tumoricidal) and CD206+ (M2 type) in cells Macrophage populations were assessed by flow cytometry for expression of IL-1, IL-1, and IL-2. Both VNP20009 and ΔppGpp were injected it-time, resulting in a 3-fold increase in the number of cells 3 days after bacterial injection. On day 1, the balance of macrophages shifted to the M1 type, and the M1 macrophages increased compared to PBS injection. The ratio of M1 to M2 macrophages (M1 / M2) increased (p<0.05, Figure 2 C) Seven days after bacterial injection, the M1 / M2 ratio was significantly higher than that of PBS-treated tumors. NP20009-injected tumors further shifted to M1 (p<0.01, Fig. 2C), whereas Δ In ppGpp-injected tumors, the M1 / M2 ratio was decreased compared to the 3-day time point, and there was no significant difference from PBS. Interestingly, the attenuated effect of it on macrophage numbers was no longer observed (Figure 2C). Injection of S. typhimurium had a systemic effect. Over the next 3 days, splenic macrophages (CD45+F4 / 80+) were cultured in PBS (15.4% ) compared with the VNP20009 group (38.8%, p<0.01) and the ΔppGpp group ( 32.4%, p<0.01) (Figure 9).

[0072] Increased tumor-infiltrating lymphocytes were observed in bacterially injected allografted schwannoma, and M Considering the shift to type 1 macrophages, the intratumoral T cell composition may be affected by attenuated S. typhimurium. We investigated whether intravital injection of S. typhimurium alters tumor invasion. Regulatory T cells (CD3 / CD4), cytotoxic T cells (CD3 / CD8) and regulatory T cells Cells (Treg, CD4 / CD25) were analyzed by multicolor flow cytometry 7 days after it bacterial injection. The CD4+ hemoglobin levels were assessed using cytometry (n=3 / group). No effect on Par T cells was observed with VNP20009 or ΔppGpp Injection of either antibody increased the expression of CD8+ cytotoxic T cells compared with PBS injection. -Percentages increased (7.56%, 7.56%, and 2.79%, respectively; Figure 2D). Furthermore, VNP2009 or ΔppGpp injection significantly increased the cytotoxicity of α- and β-glucan-containing ... and the number of tumor-infiltrating CD25+ Tregs was reduced (4.15%, 3.24%, and and 8.32%, Figure 2D). In all cases, these percentages were significantly higher than CD45 + represents the percentage of cells.

[0073] Second, they are involved in ICD and regulate the survival, proliferation, and differentiation of both immune and tumor cells. Two important immune-stimulating cytokines, tumor necrosis factor-α and alpha- tumor necrosis factor-α (TNF-α) and interferon gamma (IFN-γ) 44、45 VN against The effects of P2009 or ΔppGpp it injection were investigated. Attenuated S. typhimurium also induces a shift to M1 macrophages. Therefore, we hypothesized that bacterial infection of schwannoma induces the production of these cytokines. Furthermore, S. typhimurium showed antitumor activity (Fig. 2C). Two pro-inflammatory cytokines known to promote inflammatory responses, namely IL-1β and IL-1β, -18 19、47、48 Inflammasome (NL) is involved in the processing and maturation of (including RP3 and NLRC4)46 It is a known inducer of VNP20009- and ΔppGpp-mediated changes in the M1 / M2 ratio were evident. (Fig. 2C) and elevated levels of multiple cytokines were observed in the schwannoma allografts within the sciatic nerve. mRNA and protein were extracted from tumors and cytokine profiles were analyzed using RT-PCR. Proinflammatory cytokine T was assessed using PCR and ELISA (N=3 / group). Transcription levels of NF-α, IFN-γ, IL-1β, and IL-18 were significantly increased compared with controls. , VNP20009 and ΔppGpp S. typhimurium injection tumors Notably, VNP20009 was upregulated in tumors (Fig. 2E). Injected tumors showed higher TNF-α and IFN-γ mRNA expression compared to ΔppGpp. The observed levels were p<0.01 and p<0.001, respectively. TNF-α, IFN-γ, IL-1β, and IL-18 protein levels were was elevated in VNP20009- and ΔppGpp-injected tumors compared with PBS controls ( Figure 2F Furthermore, itVNP20009 injection significantly increased tumor size compared to ΔppGpp. In the presence of IL-18, IFN-γ, and TNF-α proteins, (p<0.05, p<0.01, p<0.05, respectively; Figure 2F). Treatment also significantly increased NLRC4 and NLRP3 expression compared to ΔppGpp or PBS. mRNA was elevated to a greater extent (p<0.01, Figure 2E).

[0074] [Example 3] It S. typhimurium VNP20009 injection, bacterial injection The growth of injected and contralateral non-injected allografted schwannoma tumors in mice is controlled. A subset of schwannomas contains CD4+ and CD8+ T cells that express PD-1. have been shown to increase the anti-tumor immunity of these cells. 49 Homologous Injection of transplanted schwannoma with itVNP20009 increased the number of CD8+ cytotoxic T cells and and CD25+ Treg numbers, suggesting activation of the adaptive immune response. Therefore, systemic anti-PD-1 monoclonal antibody (mAb) and i. In combination with t.VNP20009 injection, the host's bacteria-induced antitumor adaptive immune response was enhanced. It was evaluated that 08031-9 mouse schwannoma cells may be effective against FVB / N mice. The mice were subcutaneously implanted into both flanks of the mice and divided into four groups (Figure 3A shows the experimental design): i) itV NP20009 (left flank tumor), ii) ip anti-PD-1 mAb P, iii) i t.VNP20009 and ip anti-PD-1 mAb, and iv) itP BS (left tumor). Subcutaneous implantation was used instead of intrasciatic nerve implantation, because the former This allows for longer survival and therefore a greater opportunity for an adaptive immune response to occur. The average tumor size is approximately 150 mm 3 50 (11 days after transplantation) When the concentration reaches 100μL, add VNP20009 (10 4 CFU) or PBS into the left flank In parallel, anti-PD-1 mAb (250 μg / injection) was injected directly into the transplanted tumor. ) were injected i.p. on days 10, 13, 16, and 19 after tumor cell implantation. 51 i. t. Compared with PBS injection, either VNP20009 or anti-PD-1 mAb Monotherapy with itVNP2009 inhibited the growth of both tumors (Figure 3B). , resulted in greater growth control of uninjected tumors than PD-1-mAb (p<0.0 5, Figure 3B, D). The combination will be compared to 1) either VNP20009 or anti-PD-1 mAb alone 1) enhanced growth control of bacterial-injected tumors (p<0.05, Figure 3B); and 2) anti-PD Compared with -1mAb treatment, tumor growth control of non-injected contralateral tumors was enhanced (p<0.05 ), but there was no difference compared with VNP20009-treated mice ( Fig. 3D ).

[0075] These effects on schwannoma growth suggest that itVNP20009 may be able to suppress tumor growth. This effect is mediated by immune checkpoint inhibition, which generates an adaptive immune response that can control proliferation. This suggests that the effects of the toxic substance may be enhanced by the toxic substance. T cell subsets in these tumors, specifically helper (CD3 / C D4) T cells, cytotoxic (CD3 / CD8) T cells, and regulatory T (CD4 / CD2) 5) T cells were analyzed (Figure 3C). In the left flank tumors (Figure 3B, C), itVNP2 0009 and systemic anti-PD-1 mAb compared with 1) PBS injection (1.01%) increased CD8+ cytotoxic T cells compared with placebo (11.8% and 10.8%, respectively); 2) Increase in CD4+ helper T cells (VNP2000) compared to PBS injection (0.77%) 9 (3.43%); anti-PD-1 mAb (4.21%)), and 3) PBS (31.2 % compared with VNP20009 (15.5%); anti-PD-1 The combination of VNP20009 and anti-PD-1 mAb resulted in a significant improvement in PD-1 expression. Combined, CD8+ (24.9%) and CD4+ (31.2%) were significantly higher than either monotherapy or PBS. (29.6%) T cells increased additively (Figure 3C). 15.5%); anti-PD-1 mAb (19.3%), and PBS (31.2%) In comparison, the combination of bacteria and immune checkpoint blockade reduced Treg activity (7.59%). There was also an additive effect of the combination.

[0076] To further evaluate whether these manipulations induced a systemic host antitumor immune response, We analyzed the same T cell population in right flank tumors that did not receive bacterial injection. ItVNP20009 (left flank tumor) in combination with mAb VNP 20009 (4.55%) or anti-PD-1 mAb (3.26%) alone A synergistic effect was observed in the non-injected tumors against infiltrating CD4+ helper T cells (23.4%). Neither monotherapy was different from PBS (2.24%) (Figure 3E). The percentage of CD8+ cytotoxic T cells in the tumor was significantly increased by anti-PD-1 mAb (6 VNP20009 / anti-PD-L1 expression compared with both VNP20009 / anti-PD-L1 expression (0.05%) and PBS treatment (4.92%). with either the mAb combination (47.5%) or VNP20009 (41.9%) The VNP20009 / anti-PD-1 mAb combination increased the Finally, as shown in Figure 3E, each treatment regimen Compared with itPBS injection (in the contralateral left flank tumor), tumor-infiltrating CD25+ Decreased percentage of Tregs: 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 depletion was similar to that of VNP200. The highest efficacy was in mice with 09 / anti-PD-1 mAb, whereas VNP20009 was anti-PD-1 It had a greater effect than the mAb.

[0077] [Example 4] Primary allograft murine schwannoma infected with S. typhimurium (VN P20009) injection inhibits the growth of schwannomas rechallenged without bacterial injection. in schwannoma with itVNP20009 alone or in combination with anti-PD-1 mAb. To investigate whether a sustained anti-tumor adaptive immune response could be generated, a rechallenge model was performed. 08031-9 mouse schwannoma cells were transplanted into the left flank of FVB / N mice. The cells were transplanted and divided into the following groups as shown schematically in Figure 5A: i) itVNP20009, i i) ip anti-PD-1-mAb P, iii) itVNP20009 and ip .anti-PD-1 mAb, and iv) itPBS. Average tumor size was 150 mm 3 50 When the concentration reached 100μL (8 days after transplantation), itVNP20009 (10 in 100μL) 4 C FU) was directly injected. Anti-PD-1 mAb (250 μg / injection) 51 , tumor cell transplantation The results replicate those shown in Figure 3. and all treatment regimens, i.e., VNP20009 / anti-PD-1 mAb, VNP2 0009, and anti-PD-1 mAb inhibited tumor growth compared with PBS, and VNP2 There was an additive effect of combining 0009 with anti-PD-1 mAb (Figure 4B). 12 days after bacterial injection of sc tumors (and first application of immune checkpoint inhibitors) After 13 days of treatment, animals were transplanted with 08031-9FC schwannoma cells into the contralateral sciatic nerve. The animals were re-challenged by the same method. Therefore, these allograft schwannomas develop more rapidly intraneurally than subcutaneously, resulting in the development of sciatic nerve Both intracranial and intrasciatic nerve locations were selected. Tumor growth within the sciatic nerve was monitored via bioluminescence imaging. VNP20009 or VNP20009 / anti-PD-1 compared with PBS control. Tumor growth was inhibited in mice previously treated with the mAb, with no significant difference between the two treatments. Previous treatment with anti-PD-1 mAb alone revealed no significant difference (Figure 4D). There was no change in tumor growth compared to PBS (Figure 4D). The magnitude of the inhibitory effect of 9 was greater in the rechallenge tumors ( ) than in the primary bacterial injection schwannomas ( Figure 4B ). Figure 4D) appears to be larger.

[0078] We analyzed the injected tumors and rechallenge tumors at the time of sacrifice by flow cytometry. The T cell composition of the tumors was analyzed again. In subcutaneous primary tumors, the population of infiltrating CD4+ helper T cells was significantly higher. -Percentage of VNP20009 (8.4%) compared to PBS-injected control (0.99%) 7%), anti-PD-1 mAb (3.39%), and anti-PD-1 mAb and VNP200 09 (9.39%); VNP20009 was anti-PD1-m Although the effect of checkpoint blockade on bacteria was greater than that of Ab, the effect of bacteria alone was not significant. There was no increased effect compared to the control group (Figure 4C). CD8+ cytotoxicity in subcutaneous tumors The percentage of T cells was also significantly higher in VNP20009 ( 11.2%), anti-PD-1 mAb (6.69%), and VNP20009 and anti-PD- 1 mAb (15.5%); in this case, the immune checkpoint inhibitor The increase was greater in bacterial-injected tumors than in intravenously treated tumors, and the combination therapy increased CD more than bacteria alone. In contrast, induction of 8+ T cells was significant (Figure 4C) compared to the PBS control (12.2%). The percentage of CD25+ regulatory T cells in subcutaneous tumors was significantly increased by systemic anti-PD-1 mAb ( 11.2%), but not with itVNP20009 injection (9.52%). and decreased by both bacterial injection and checkpoint inhibition in combination (4.90%; (Figure 4C). This suppressive effect of the combination therapy on tumor Tregs was greater than that of bacterial treatment alone. was also large (Fig. 4C).

[0079] Quantification of T lymphocyte populations in intrasciatic nerve rechallenge tumors compared with primary subcutaneous tumors A different pattern of treatment effect was evident. The percentage of CD4+ helper T cells was significantly increased by VNP20009 and anti-PD-1 mAb. b (13%); Efficacy of NP20009 (7.49%) or anti-PD-1 mAb monotherapy (6.93%) In these rechallenge tumors, there was no significant difference compared to the PBS control (11.5%) (Figure 4E). Compared with VNP20009 (14.1%) or VNP20009 / anti-PD-1 mAb The percentage of CD8+ cytotoxic T cells in mice previously treated with the combination (23.1%) previous checkpoint blockade alone was ineffective (anti-PD-1 mAb, 12.2%; Figure 4E). Similarly, CD25+ Tregs in rechallenge tumors The percentage of VNP200 mice with HIV-1 infection was significantly higher than that of PBS control mice (10.4%). 09 (6.01%) or VNP20009 / anti-PD-1 mAb combination (3.82%) Treatment with anti-PD-1 mAb (8.16%) reduced the PD-1 expression, but not with anti-PD-1 mAb (8.16%) ( Figure 4 E) Effect of previous immune checkpoint on Treg percentage in rechallenged schwannoma. Although cross-link inhibition alone had no effect, the addition of anti-PD-1 mAb to VNP20009 The addition of the β-lactam serovar 1000 enhanced the inhibitory effect of the previous bacterial treatment alone (Fig. 4E).

[0080] [Example 5] Allograft mouse schwannoma by S. typhimurium injection Inhibition of angiogenesis S. typhimurium inhibits endothelial proliferation, an important pro-angiogenic factor. It reduces the expression of VEGF (vascular endothelial growth factor) 52 , which demonstrated anti-angiogenic properties in preclinical cancer models It has been shown to have 24、53 Anti-angiogenic monoclonal antibody against VEGF-A The antibody bevacizumab inhibits schwannoma growth in a subset of individuals with schwannoma. Can be controlled 54 Given these observations, the role of attenuated S. The efficacy of intravenous injection of S. typhimurium was investigated.

[0081] Intrasciatic mouse 08031-9 schwannoma was inoculated with attenuated S. typhimurium (S. typhimurium). The tumor vasculature was assessed 2 weeks after intravenous injection of 1000 mg / kg of tetracycline (T2), which was associated with increased tumor vascularity compared to PBS-injected controls. Tumor angiogenesis was directly visualized by the endothelial marker CD31+. and assessed by immunohistochemistry 55 (Figure 5). Macroscopic evaluation of tumors (N=6 / mouse) group) are pale in color and have minimal or no external vascularization. All tumors were bright red and had prominent external vascularity compared to S. typhimurium-injected tumors. The difference between the PBS-injected and PBS-injected schwannomas was easily discernible (Figure 5). Both the PBS-injected tumors and the ΔppGpp-injected tumors were significantly higher than the PBS-injected tumors (45.71 ± 4.75 ; Figure 5, N = 3 / group), the number of CD31+ cells was reduced (7.71 ± 0.01, respectively) compared with the control group. 1.89, P<0.001 and 8.41±1.68, P<0.001).

[0082] [Example 6] Injection of S. typhimurium into xenografted human NF1, The growth of human sporadic MPNSTs and human meningioma subcutaneous tumors is suppressed. Human NF1-related (S462TY, Fig. 6A) or or sporadic (STS26, Figure 6B) malignant peripheral nerve sheath tumor (MPNST) cells were subcutaneously implanted. did 56 on the growth of the human NF-1 xenograft model. The effects of injection of VNP20009 and ΔppGpp were also evaluated. Furthermore, the efficacy of itS S. typhimurium was evaluated in nu / nu mice. Benign meningioma (Ben-Men-1, Fig. 6C) or malignant meningioma (CH-157 MN, Fig. 6D) This was demonstrated in a subcutaneous xenograft model in which a cell line was implanted. The samples were divided into three groups (n=5): VNP20009 or ΔppGpp (10 in 100 μl) 4 C FU) versus PBS-injected control. Intratumoral injections were performed once tumor masses were visible to the naked eye. Tumor growth was monitored by caliper measurements.

[0083] In the NF-1 xenograft model, our data showed that VN compared with itPBS injection i.t. injection with either P20009 or ΔppGpp inhibited NF-1-associated The tumor growth of S462TY MPNST cells was significantly suppressed (P<0.001, Figure 6A). , tumor growth of rapidly growing sporadic STS26T MPNST cells was suppressed (P<0 At the time of sacrifice (day 31 and day 4 for S462TY), STS26T) and PBS control (day 30 for VNP20009 and and ΔppGpp-injected mice in the S462TY and STS26T models, respectively. The tumor sizes were seven times smaller and four times smaller.

[0084] Similarly, in a meningioma xenograft model, either VNP20009 or ΔppGpp injection of β-lactamase inhibitors (Ben-Men-1, Figure 6C) compared to PBS controls. There was a significant regression of meningioma tumor growth, and the tumor growth of malignant (CH-157, Figure 6D) meningioma was suppressed. In a benign model, VNP20009 S. typhimurium or ΔppGpp S. typhimurium In mice, on the day of sacrifice (day 38), 2 out of 5 mice showed complete regression. On the day of sacrifice of malignant meningiomas, VNP20009 and ΔppGpp were significantly increased compared to the PBS control. In the injected mice, the tumor size was approximately one-third that of the control group. The experiment was stopped on day 23.

[0085] References

[0086] [Table 2-1]

[0087] [Table 2-2]

[0088] [Table 2-3]

[0089] [Table 2-4]

[0090] [Table 2-5]

[0091] [Table 2-6]

[0092] [Table 2-7]

[0093] [Table 2-8]

[0094] [Table 2-9]

[0095] Other embodiments The present invention has been described in conjunction with the detailed description thereof, and the foregoing description provides the principles of the present invention as defined in the appended claims. The scope of the invention as defined by the ranges is intended to be illustrative and not limiting. It should be understood that other aspects, advantages, and modifications are within the scope of the following claims. be.

Claims

1. A method of treating a subject having or at risk of having a benign nervous system tumor, comprising administering to the subject optionally in combination with immune checkpoint inhibitors and / or anti-angiogenic agents. administering to the subject a therapeutically effective amount of a composition comprising attenuated Salmonella bacteria. and

2. The subject is a patient with neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); sis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis ( benign tumors or tumor-related tumors selected from the group consisting of:

10. The method of claim 1, wherein the subject has or has been diagnosed as having a related condition.

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

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

5. The conditions associated with an increased risk of benign nervous system tumors include neurofibromatosis 1 (NF1); 5. The method of claim 4, wherein the disease is fibromatosis 2 (NF2); or schwannomatosis.

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

7. The attenuated Salmonella is an attenuated form of S. typhimurium.

7. The method according to any one of claims 1 to 6, wherein the bacterial strain is a toxigenic strain.

8. The attenuated strain of S. typhimurium contains modified lipid A (msbB -) and Salmonella enterica serovars with purine auxotrophic mutation (purI-) Salmonella enterica serovar typhimurium strain VNP2000 9. The method of claim 7 .

9. 10. The method of claim 9, wherein the composition is free of Clostridium novyi.

1. The method according to claim 1.

10. The attenuated Salmonella is an intracellularly inducible Salmonella protease. The method of claim 1, which does not contain a lysis gene or cassette operably linked to a motor. How to do it.

11. The checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling. The method of claim 1 .

12. The inhibitor of PD-1 signaling is selected from the group consisting of PD-1, CD40, PD-L1, and CT. The method according to claim 11, wherein the antibody is an antibody that binds to LA-4.

13. The angiogenesis inhibitor is an angiogenesis inhibitor that inhibits vascular endothelial growth factor (VEGF) or its receptor (VEGFR 2. The method of claim 1, wherein the compound is an inhibitor of .

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

15. Use in methods of treating subjects having or at risk of having benign nervous system tumors for the treatment of rheumatoid arthritis, optionally in combination with checkpoint inhibitors and / or anti-angiogenic agents A composition comprising live attenuated Salmonella bacteria.

16. The subject is a patient with neurofibromatosis 1 (NF1); neurofibromatosis 2 (NF2); sis; meningioma; schwannoma; vestibular schwannoma; sporadic schwannoma; neurofibroma; neurofibromatosis ( benign tumors or tumor-related tumors selected from the group consisting of:

16. The method of claim 15, wherein the subject has or has been diagnosed as having a related condition. Composition of.

17. 16. The composition for use according to claim 15, wherein the subject does not have a malignant solid tumor.

18. 16. The method of claim 15, wherein the subject has a condition associated with an increased risk of benign nervous system tumors. The composition for use as described above.

19. The conditions associated with an increased risk of benign nervous system tumors include neurofibromatosis 1 (NF1); 19. The use of claim 18, wherein the disease is fibromatosis 2 (NF2); or schwannomatosis. Composition for.

20. The attenuated Salmonella is formulated for intratumoral or intravenous administration.

16. The composition for use according to claim 15, wherein the composition is hydroxylated.

21. The attenuated Salmonella is an attenuated form of S. typhimurium.

21. A composition for use according to any one of claims 15 to 20, which is a toxigenic strain.

22. The attenuated strain of S. typhimurium contains modified lipid A (msbB -) and Salmonella enterica serovars with purine auxotrophic mutation (purI-) Salmonella enterica serovar typhimurium strain VNP2000 22. The composition for use according to claim 21, wherein the hydroxyl group is 9.

23. 10. The method of claim 9, wherein the composition is free of Clostridium novyi.

16. A composition for use according to 15.

24. The attenuated Salmonella is an intracellularly inducible Salmonella protease.

16. The method of claim 15, which does not include a lysis gene or cassette operably linked to a motor. The composition for use as described above.

25. The checkpoint inhibitor is an inhibitor of PD-1 or CTLA-4 signaling.

16. A composition for use according to claim 15, wherein

26. The inhibitor of PD-1 or CTLA-4 signaling is selected from the group consisting of PD-1, CD40, PD 26. The composition for use according to claim 25, wherein the antibody is an antibody that binds to CTLA-L1 or CTLA-4. Finished product.

27. The angiogenesis inhibitor is an angiogenesis inhibitor that inhibits vascular endothelial growth factor (VEGF) or its receptor (VEGFR 16. The composition for said use according to claim 15, wherein the compound is an inhibitor of .

28. 16. The composition for use according to claim 15, wherein said inhibitor of VEGF is bevacizumab. 。

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