Treatment of benign nervous system tumors using attenuated salmonella typhimurium

Abstract

Compositions and methods for treating benign nervous system tumors, including schwannomas, using attenuated salmonella typhimurium and optionally one or more checkpoint inhibitors.

Description

[0001] Priority Statement

[0002] This application claims the benefit of U.S. Patent Application Serial No. 62 / 811,066, filed February 27, 2019. The entire contents of the foregoing are incorporated herein by reference. Technical Field

[0003] This article provides compositions and methods for treating benign nervous system tumors, including schwannomas, using attenuated Salmonella typhimurium and optionally one or more checkpoint inhibitors. Background Technology

[0004] Schwannomas are slow-growing, benign growths originating from Schwann-lineage cells. 1,2 Depending on their location and size, these tumors can cause a wide range of neurological deficits, including hearing loss, imbalance, tinnitus, motor loss, and severe pain. 3,4 In some cases, they can die due to brainstem compression. 5 Schwannomas can occur sporadically (hence the term "sporadic schwannomas") or as part of neurofibromatosis type 2 (NF2) and schwannomatosis, debilitating genetic syndromes. 6 Treatment for schwannomas is primarily limited to surgical resection and symptom management of pain. Resection is often non-curable for many patients, frequently associated with additional nerve damage, and may be impractical due to the location or large number of tumors. 7 The efficacy of anticancer therapies for schwannomas has not yet been proven, due to the slow-replicating nature of these benign lesions. 8,9,10 Bevacizumab is currently the only widely accepted drug therapy for schwannomas; it temporarily stabilizes tumor growth by targeting the highly vascularized nature of these neoplasm subpopulations. 8,9,11 Unfortunately, current pain management strategies are insufficient for many people, further increasing the burden of the disease. The fact that schwannomas can occur in multiple locations and new lesions can develop throughout a person's life complicates treatment. Therefore, schwannomas and related diseases cause lifelong suffering that cannot be stably controlled with current treatment options. Summary of the Invention

[0005] Schwannomas are slow-growing benign growths that occur anywhere in the body, including along the spinal cord and within the skull. Schwannomas often first appear in childhood or adolescence, with new tumors developing throughout life. These tumors cause pain, sensory / motor dysfunction, and death by compressing peripheral nerves, the spinal cord, and / or the brain. The immense suffering and debilitating effects associated with schwannomas, coupled with the lack of treatment options, make their treatment a major unmet medical need. This article describes a therapeutic approach involving intratumoral (it) injection of attenuated Salmonella typhimurium for benign growths including schwannomas. Current results demonstrate the ability of this intratumoral Salmonella typhimurium to control tumor growth in both xenografted human NF2 schwannomas models in nude mice and allogeneic genetic mouse schwannomas models in immunocompetent animals. Schwannoma growth control in the allogeneic transplantation models was associated with tumor cell apoptosis, reduced tumor angiogenesis, and the induction of antitumor adaptive immune responses. Intratumoral injection of *Salmonella typhimurium* resulted in tumor control of both the injected tumor and concurrently developing distal schwannomas. Furthermore, 13 days after initial treatment, intratumoral *Salmonella typhimurium* controlled the growth of re-challenge schwannomas implanted contralaterally to the primary tumor. In an allogeneic schwannoma model, systemic administration of a programmed death-1 receptor (PD-1) checkpoint inhibitor controlled tumor growth to the same extent as intratumoral *Salmonella typhimurium*, and the combination of these two therapies had an additive effect on growth control in injected tumors and a synergistic effect on T-cell subsets.

[0006] Current data support the use of intratumorally attenuated Salmonella typhimurium in combination with PD-1 checkpoint inhibition as an immunotherapy capable of controlling the growth of both injected and uninjected benign nervous system tumors, including schwannomas and schwannomas-associated vegetations (including NF1-associated tumors and meningiomas). The data provided further demonstrate the potential of this treatment strategy to control tumor growth following initial treatment. Importantly, direct injection of attenuated Salmonella typhimurium into the tumor exhibits a vaccine-like effect, inducing an antitumor adaptive immune response. These results represent the first reported application of bacterial tumor therapy for benign vegetations and the first demonstration of immunotherapy for schwannomas.

[0007] Therefore, this article provides a method for treating subjects with benign nervous system tumors or at risk of developing benign nervous system tumors. The method includes administering to the subject a therapeutically effective amount of a composition comprising, optionally in combination with an immune checkpoint inhibitor and / or angiogenesis inhibitor, live attenuated Salmonella. Furthermore, this article provides a method for treating subjects with benign nervous system tumors or at risk of developing benign nervous system tumors using a composition comprising, optionally in combination with a checkpoint inhibitor and / or angiogenesis inhibitor.

[0008] In some implementations, the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the following groups: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), schwannomatosis, meningioma, schwannoma, vestibular schwannoma, sporadic schwannoma, neurofibroma, neurofibromatosis (NF), or any combination thereof. In some implementations, the subject does not have a malignant solid tumor (i.e., has not been diagnosed with a malignant solid tumor). In some implementations, the subject has a condition associated with an increased risk of benign nervous system tumors, such as neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), or schwannomatosis.

[0009] In some implementations, attenuated Salmonella is administered intratumorally or intravenously.

[0010] In some implementations, the attenuated Salmonella is an attenuated strain of Salmonella typhimurium, such as Salmonella enterica serovar typhimurium strain VNP20009, which has modified lipid A (msbB–) and purine auxotrophic mutation (purI–).

[0011] In some embodiments, the composition does not contain Clostridium novyi.

[0012] In some implementations, the attenuated Salmonella does not contain a cleavage gene or cassette that is operatively linked to an intracellularly induced Salmonella promoter.

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

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

[0015] Furthermore, this document provides a method for treating benign schwannoma in mammals, the method comprising administering to the mammal a therapeutically effective dose or titer of an attenuated strain of pathogenic enteric bacteria. In some embodiments, the attenuated strain of pathogenic enteric bacteria is *Salmonella typhimurium*. In some embodiments, the attenuated strain of *Salmonella typhimurium* has deletions of the pull and msbB genes, the strain being named VNP20009. In some embodiments, the attenuated strain of *Salmonella typhimurium* is defective in guanosine 5'-diphosphate-3'-diphosphate synthesis, the strain being named 8ppGpp. In some embodiments, administration includes, but is not limited to, intravenous injection or direct injection into the benign schwannoma. In some embodiments, the schwannoma includes, but is not limited to, neurofibroma or schwannoma. In some embodiments, the tumor includes, but is not limited to, those associated with neurofibromatosis type 1, neurofibromatosis type 2, schwannoma, or sporadic schwannomas.

[0016] In some embodiments, the method includes administering a therapeutically effective dose of an attenuated strain of pathogenic intestinal bacteria and a checkpoint inhibitor to the mammal. In some embodiments, the checkpoint inhibitor includes, but is not limited to, peptides, antibodies, small molecules, microRNAs, antisense oligonucleotides, or small interfering RNAs. In some embodiments, the checkpoint inhibitor is a monoclonal antibody that binds to an epitope of an antigen. In some embodiments, the epitope binding to the monoclonal antibody is in the PD-1 or CTLA-4 antigen.

[0017] In some implementations, the mammal is human.

[0018] Furthermore, this document provides pharmaceutical compositions comprising an attenuated strain of pathogenic enteric bacteria in a pharmaceutically acceptable carrier, and optionally a checkpoint inhibitor. In some embodiments, the attenuated strain of pathogenic enteric bacteria is *Salmonella typhimurium*.

[0019] In some implementations, the checkpoint inhibitor is a monoclonal antibody. In some implementations, the monoclonal antibody binds to an epitope of the PD-1 or CTLA-4 antigen.

[0020] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Methods and materials used in this invention are described herein; other suitable methods and materials known in the art may also be used. Materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification, including the definitions, shall prevail.

[0021] Other features and advantages of the invention will be apparent from the following detailed description and drawings, as well as from the claims. Attached Figure Description

[0022] Figure 1A -D. Intratumoral injection of attenuated Salmonella Typhimurium controlled schwannoma development in a human HEI-193 xenograft schwannoma model and a mouse 08031-9 allogeneic schwannoma model. A) Intratumoral injection of VNP20009 and ΔppGpp induced significant tumor regression 2 weeks after tumor cell transplantation (n = 8 mice / group). B) Injection of VNP20009, but not ΔppGpp, at 1 week after tumor cell transplantation resulted in growth control (n = 8 mice / group). Intratumoral injection of VNP20009 and ΔppGpp resulted in increased apoptosis in both the HEI-193 xenograft model (C) and the 08031-9 allogeneic schwannoma model (D) compared to a PBS-injected control (n = 3 mice / group, yellow arrows indicate representative apoptotic bodies). Repeated-measures ANOVA was used to compare tumor signal between groups, and one-way ANOVA was used for apoptotic body analysis. Data are expressed as mean ± SEM. *p<0.05, **p<0.01. ***p<0.001.

[0023] Figure 2A -F. Intra-ischial allogeneic schwannoma injection with Salmonella typhimurium increased pro-inflammatory cytokines and altered immune cell infiltration. (A) Compared with tumors injected with PBS, mouse allogeneic schwannoma models injected with Salmonella typhimurium showed increased immune cell infiltration (CD45). + Normal white blood cells and CD68 + Pan macrophages (yellow arrows indicate positive staining (n = 3 mice / group)). (B) CD45 + Cells (left) and CD68 + Cell quantification (right) showed a significant increase in leukocytes and macrophages in tumors of mice injected with VNP20009 and ΔppGpp compared to the PBS control. (C) Identified as CD45+ F4 / 80 + Flow cytometry analysis of tumor-associated macrophage subpopulations. CD86 expression was used to identify M1 macrophages, while CD206 expression was used to identify M2 macrophages. The M1 / M2 ratio was calculated using CD45+. + F4 / 80 + %M1(CD68) + Group divided by CD45 + +F4 / 80 + %M2(CD206) + (D) Population. M1 / M2 ratio of TAM on day 3 (left) and day 7 (right). (D) Adaptive immune cell infiltration of injected tumors by flow cytometry at day 7 post-bacterial injection. Percentages shown represent CD4+. + T cells (CD3) + CD4 + CD8 + T cells (CD3) + CD8 + ) and CD25 + T cells (CD4) + CD25 + Flow cytometry is shown in Figure 10. Quantitative RT-PCR (E) and ELISA (F) of cytokines and inflammasomes in the tumor microenvironment 3 days after Salmonella Typhimurium injection. One-way ANOVA was used to compare different treatments. Data are presented as mean ± SEM. N = 3 / group. An asterisk (*) indicates a difference compared to the PBS control; a hash (#) indicates a difference compared to ΔppGpp. * / #p<0.05, ** / ##p<0.01. *** / ###p<0.001.

[0024] Figure 3A -E. Intratumoral injection of VNP20009 inhibited tumor growth in both treatment-naïve and untreated distal schwannomas, and the addition of systemic anti-PD-1 mAb enhanced killing of primary tumors. A) Experimental protocol using combination therapy with VNP20009 and anti-PD-1 mAb. B) Bilateral subcutaneous implantation of 08031-9 cells in FVB / n mice (n = 6 mice / group). Significant tumor regression was observed in mouse tumors treated with either anti-PD-1 mAb or VNP20009 monotherapy compared to PBS. The combination therapy resulted in more significant tumor regression compared to either VNP20009 or anti-PD-1 mAb monotherapy. C) Tumor sites were collected at the end of the study, and cytotoxic CD8+ was analyzed by flow cytometry. + T cells, helper CD4 + T cells and regulatory CD25+ T cells (N=3 / group). D) Although anti-PD-1 mAb significantly reduced tumor growth at uninjected sites compared to PBS, VNP20009 monotherapy and VNP20009 / anti-PD-1 mAb combination therapy significantly enhanced tumor growth control at uninjected sites compared to anti-PD-1 mAb or PBS treatment groups. E) Uninjected tumor sites were collected on day 26 post-implantation and cytotoxic CD8+ was analyzed by flow cytometry. + T cells, helper CD4 + T cells and regulatory CD25 + T cells (N=3 / group). Flow cytometry was performed on... Figure 9 As shown in the figure. 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 expressed as mean ± SEM. *p<0.05, **p<0.01.

[0025] Figure 4A -E. Intratumoral injection of VNP20009 inhibited tumor growth in experimental schwannomas injected and subsequently implanted, and the addition of systemic anti-PD-1 mAb enhanced the killing effect on injected tumors. A) Experimental protocol using combination therapy of VNP20009 and anti-PD-1 mAb. B) Subcutaneous implantation of 08031-9 cells (n=6 / group) into the left side of FVB / n mice. Significant tumor regression was observed in mouse tumors injected with either anti-PD-1 mAb or VNP20009 monotherapy compared to PBS. The combination therapy resulted in more significant tumor regression compared to monotherapy with either VNP20009 or anti-PD-1 mAb. C) Tumor sites were collected at the end of the study, and cytotoxic CD8 was analyzed by flow cytometry. + T cells, helper CD4 + T cells and regulatory CD25 + T cells. D) On day 20 post-implantation, 08031-9FC tumor cells expressing firefly luciferase were implanted into the distal sciatic nerve of mice that had received the initial injection (n=5 / group), and the tumors were monitored by bioluminescence. E) On day 16 post-implantation, secondary tumor sites were collected and cytotoxic CD8+ cells were analyzed by flow cytometry. + T cells, helper CD4 + T cells and regulatory CD25 + T cells. Flow cytometry plots are shown in Figure 10. Repeated-measures ANOVA was used to analyze tumor volume and bioluminescence signal. One-way ANOVA was used to analyze flow cytometry data. Data are presented as mean ± SEM. *p<0.05, **p<0.01.

[0026] Figure 5 Intratumoral injection of *Salmonella typhimurium* inhibited angiogenesis in intraisical allogeneic mouse schwannomas. As determined by CD31+ staining and direct observation, tumors injected with VNP20009 and ΔppGpp, collected 2 weeks after bacterial injection, showed reduced angiogenesis compared to PBS controls. Immunohistochemistry based on 3 tumors / groups shows representative staining, with red arrows indicating positive endothelial cells. Image-J was used to analyze CD31+ staining. + Cell profiling was performed for quantification, and one-way ANOVA was used for data analysis. Data are presented as mean ± SEM. ***p<0.001.

[0027] Figure 6A -D. Intratumoral injection of Salmonella typhimurium inhibited the growth of xenograft human NF1, sporadic human MPNST, and subcutaneous human meningioma tumors. Intratumoral injection of VNP20009 or ΔppGpp induced regression of NF-1-associated (S462TY, A) malignant peripheral nerve sheath tumors (MPNST) and growth control of sporadic (STS26T, B) malignant peripheral nerve sheath tumors (MPNST) compared to tumors injected with PBS. Similarly, intratumoral injection of VNP20009 or ΔppGpp resulted in regression of benign Ben-Men-1 meningioma (C) and growth control of malignant meningioma CH-157 (D) tumors compared to tumors injected with PBS. Arrows indicate the time of bacterial / PBS injection. Repeated-measures ANOVA was used to compare tumor size between groups. N = 5 mice / group. Data are presented as mean ± SEM. ***p < 0.001 (PBS compared to any Salmonella strain).

[0028] Figure 7A -B. Intratumoral injection of attenuated Salmonella Typhimurium (VNP20009) controls schwannoma development in human HEI-193 xenograft and mouse 08031-9 allogeneic schwannoma models. A) Intratumoral (it) injection of VNP20009 induced significant tumor regression in xenograft schwannoma models 2 weeks after tumor cell transplantation (n = 8 mice / group). B) VNP20009 injection in allogeneic schwannoma models 1 week after tumor cell transplantation resulted in growth control (n = 8 mice / group). Repeated-measures ANOVA was used to compare tumor signal between groups. Data are presented as mean ± SEM. *p<0.05, **p<0.01. ***p<0.001.

[0029] Figure 8Neurons burdened with Salmonella typhimurium after injection showed increased and altered immune cell infiltration. Compared with tumors injected with PBS, neurons in a human xenograft model injected with Salmonella typhimurium showed increased immune cell infiltration (CD45). + Normal white blood cells and CD68 + Pan-macrophages), (yellow arrows indicate positive staining (n=3 mice / group).

[0030] Figure 9 Flow cytometry analysis of splenic macrophages in mice injected with either Salmonella Typhimurium or PBS. Three days post-injection, immunocompetent schwannomas induced by the bacteria showed an increased macrophage population in the spleen of the injected mice. Compared to PBS (15.4%), CD45... + F4 / 80 + Flow cytometry analysis of macrophages showed an increase in macrophages in the spleen of mice injected with VNP20009 (38.8%) and in the spleen of mice injected with ΔppGpp (32.4%).

[0031] Figure 10A -C. Flow cytometry analysis of tumor-infiltrating immune cells in tumors of mice injected with Salmonella Typhimurium or PBS. M-1 macrophages (F4 / 80) collected from tumors 3 days (A) or 7 days (B) after bacterial or PBS injection. + CD86 + ) and M-2 macrophages (F4 / 80 + CD206 + (C) Analysis of CD4 in treated mice 7 days after injection of bacterial strains or PBS. + T cells (CD3) + CD4 + ), CD8+ T cells (CD3) + CD8 + ) and CD25 + T cells (CD4) + CD25 + )analyze.

[0032] Figure 11A -B, Flow cytometry images of tumor-infiltrating immune cells in tumors of mice injected with VNP20009, PD-1 mAb, VNP20009 / PD-1 mAb, or PBS. Analysis of T lymphocyte infiltration in tumors at the injection site (A) and the non-injection site (B). Tumors were collected 26 days post-injection and CD4+ analysis was performed. + T cells (CD3) + CD4 + CD8 +T cells (CD3) + CD8 + ) and CD25 + T cells (CD4) + CD25 + )dyeing.

[0033] Figure 12A -B. Flow cytometry images of tumor-infiltrating immune cells in tumors of mice injected with VNP20009, PD-1 mAb, VNP20009 / PD-1 mAb, or PBS. Analysis of T lymphocyte infiltration in tumors at the injection site (A) and secondary tumor sites (B). Tumors were collected at the primary tumor site 20 days post-implantation and at the secondary tumor site 16 days post-implantation for CD44 analysis. + T cells (CD3) + CD4 + CD8 + T cells (CD3) + CD8 + ) and CD25 + T cells (CD4) + CD25 + )dyeing.

[0034] Figure 13A -B. Invasiveness assays of attenuated Salmonella Typhimurium in cultured macrophage and schwannoma cell lines. (A) Representative photograph of the HEI-193 internalized Salmonella strain streaked on an agar plate 16 hours later. (B) Quantitative analysis of invasiveness as a percentage of infection shows that VNP20009 is more invasive than ΔppGpp but less invasive than wild-type Salmonella Typhimurium. The invasion efficiency of wild-type Salmonella Typhimurium in mouse macrophages, human HEI-193, and mouse 08031-9 cells was approximately 100%, 96%, and 58%, respectively. VNP20009 and ΔppGpp showed significantly lower invasion rates compared to wild-type bacteria. The infection percentages of VNP20009 in mouse macrophages, human HEI-193 cells, and mouse 08031-9 cells were approximately 38%, 23%, and 15%, respectively, while ΔppGpp showed further reduced invasiveness, with an infection percentage of 18% in mouse macrophages, 10% in HEI-193 cells, and 5% in 08031-9 cells.

[0035] Figure 14A-B. ELISA of cytokines after exposing cultured human schwannoma (HEI-193, A) or mouse schwannoma (08031-9, B) cell lines to Salmonella Typhimurium. Quantification of IL-1β, IL-18, and TNF-α by ELISA after incubation with VNP20009 or ΔppGpp showed no difference in the release of cytokines compared to PBS-treated cells. One-way ANOVA was used for intergroup comparisons. N = 3 independent experiments. Data are presented as mean ± SEM.

[0036] Figure 15A -B. ELISA of cytokines after exposing cultured human (THP-1 differentiated macrophages, A) or mouse (RAW264.7 macrophages, B) cell lines to Salmonella Typhimurium. Quantification of IL-1β, IL-18, and TNF-α by ELISA showed significant differences in the release of cytokines after incubation with VNP20009 or ΔppGpp compared to PBS-treated cells. One-way ANOVA was used for intergroup comparisons. Data are presented as mean ± SEM. N = 3 independent experiments; *p < 0.05; **p < 0.01.

[0037] Figure 16 Salmonella typhimurium did not induce cytokine release in the serum of mice with tumors in an allogeneic, immunocompetent schwannoma mouse model. Cytokine levels in the serum of injected mice were measured by ELISA 3 days after bacterial injection. One-way ANOVA was used for intergroup comparisons. Data are presented as mean ± SEM. N = 3 independent experiments. Detailed Implementation

[0038] Bacterial-mediated cancer therapy (BCT) using Gram-negative bacteria was introduced by William Coley in the mid-19th century, when he used live Streptococcus pyogenes to treat solid tumors. 12 The basic principle of bacterial cancer therapy is based on certain bacterial strains, including the Gram-negative bacterium *Salmonella typhimurium*. 13-21 It can specifically home to and proliferate in the hypoxic regions of angiogenic tumors, inducing direct lysis of tumor cells and the establishment of anti-tumor immune responses. 22 Furthermore, bacterial injection into tumors has been shown to be anti-angiogenic. 23,24Therefore, in addition to directly inducing cancer cell death, bacteria can be used as immuno-oncology and anti-angiogenic agents to target highly vascularized tumors and establish immune controls to prevent the development of new tumors.

[0039] A large amount of preclinical and clinical data supports BCT as an immunotherapy strategy. 20,25,32,57,58 Furthermore, for forty years, the intravesical application of live attenuated strains of Mycobacterium bovis has been the only FDA-approved treatment for orthotopic bladder cancer. 29 BCT using attenuated strains of Salmonella typhimurium has shown clear efficacy in several preclinical cancer models. 16,18-20 Early clinical trials of attenuated Salmonella Typhimurium-based BCTs, delivered intravenously, directly intratumorally, or orally, have demonstrated safety but failed to show efficacy. 25,26,27,28 This lack of efficacy may be due to the rapid division of cancer cells and the use of intravenous delivery. Bacterial inoculants are limited by the toxicity of systemic delivery, and in these trials, the lack of efficacy may be dose-related. Currently, there is a live attenuated strain of BCT, Mycobacterium bovis, approved by the U.S. Food and Drug Administration, which has been the standard of care for high-risk non-muscle-invasive bladder cancer for the past four decades. 29 .

[0040] However, BCT has never been recommended as a potential treatment for benign growths, possibly because benign tumors tend to be immunologically cold. 66,67 Therefore, bacterial therapy has never been tested in the context of slow-growing benign tumors such as schwannomas, where traditional cancer therapies primarily targeting highly replicating cells are ineffective. This article presents a preclinical study supporting bacterial therapy for schwannomas (benign growths of the peripheral nervous system). It is hypothesized that intratumoral injection of attenuated Salmonella typhimurium has the potential to directly kill schwannomas cells, inhibit angiogenesis, and transform the tumor immune microenvironment from relatively 'cold' to 'hot'. Further hypothesizing that the combination of immune cell death (if it occurs), the generation of a pro-immunogenic tumor environment, and VEGF / angiogenesis inhibition can synergistically generate an adaptive antitumor immune response.

[0041] To test these hypotheses, the effects of two attenuated Salmonella Typhimurium strains (VNP20009 and ΔppGpp) were evaluated in both a xenograft human NF2 model in nude mice and an allogeneic schwannoma model in FVB / N mice with syngeneic immunity. Data showed that intratumoral injection of attenuated Salmonella Typhimurium controlled schwannoma growth in both models. Intratumoral injection of Salmonella Typhimurium into schwannomas led to tumor cell killing and induced a systemic antitumor adaptive immune response in immunocompetent mice. This antitumor immune response controlled the growth of tumors not injected with bacteria during bacterial therapy and prevented the development of "rechallenge" tumors after treatment. Salmonella Typhimurium was effective in both injected and uninjected contralateral tumors, as well as in rechallenge tumors (except for those with CD4+). + (Besides the role of cells) In allogeneic schwannomas, CD4+ increases tumor invasiveness. + Helper T cells and CD8 + Cytotoxic T cells increase and CD25 levels rise. + The reduction of Tregs further supports the existence of an anti-tumor adaptive immune response. Intratumoral injection of Salmonella typhimurium increases systemic PD-1 immune checkpoint inhibition, enhancing control of schwannomas injected with the bacteria and from the contralateral uninjected tumor, rather than re-engaging the tumor. Studies of tumor-infiltrating lymphocytes (TILs) demonstrate increased CD4 counts in schwannomas injected with attenuated Salmonella typhimurium. + Helper T cells and CD8 + The number of cytotoxic T cells increased and CD25 + The number of regulatory T cells is reduced.

[0042] This study tested the ability of two attenuated Salmonella Typhimurium strains, VNP20009 and ΔppGpp, to inhibit the growth of schwannomas. In both strains, attenuation reduced the likelihood of pathogenicity, including septic shock. In vitro evaluation showed that VNP20009 was more invasive than ΔppGpp in both cultured macrophages and schwannoma cell lines, but less invasive than wild-type Salmonella Typhimurium. Figure 13A &B). Although exposure of cultured macrophages to both strains of Salmonella Typhimurium led to the release of inflammatory cytokines, co-culture of VNP20009 or ΔppGpp with schwannoma cell lines did not induce any cytokine release. Figure 14A &B and Figure 15A &B). This suggests that bacterial-macrophage interactions may play a key role in the observed antitumor effects of Salmonella typhimurium.

[0043] In vivo data showed that intratumoral (it) injection of VNP20009 or ΔppGpp monotherapy in our human NF2 xenograft model resulted in tumor regression, with no difference in the magnitude of regression between the two tested strains. Furthermore, in an immunocompetent mouse allogeneic schwannoma model, intratumoral injection of VNP20009 controlled tumor growth. The therapeutic efficacy of VNP20009 was demonstrated by an increase in apoptotic bodies in the tumor microenvironment compared to ΔppGpp or PBS. Figure 1D ) and increased release of inflammatory cytokines including IL-18, TNF-α, and IFN-γ. Figure 2E &F) was used for monitoring. No increased systemic cytokine levels were observed in mice injected with Salmonella typhimurium. Figure 16 Further research focused on analyzing immune profile alterations in the tumor microenvironment of immune schwannomas models that had received intratumoral injection of VNP20009 bacteria or PBS.

[0044] M2 macrophages and myeloid-derived suppressor cells (MDSCs) have been shown to infiltrate vestibular schwannomas and are associated with progressive tumor growth. 42,59 Unlike M2 macrophages, which promote tumor growth, M1 macrophages have immunostimulatory effects and inhibit tumor growth, and at least partially contribute to an adaptive immune response through phagocytosis and antigen presentation. 60-65 In an allogeneic schwannoma model, by day 3 post-bacterial injection, intratumoral VNP20009 injection, which controls tumor growth, reduced CD45 levels. + F4 / 80 + The ratio of M1 macrophages to M2 macrophages is increased in tumor-activated macrophages (TAMs).

[0045] Treatment

[0046] As shown in this article, VNP20009 is an attenuated strain of Salmonella, such as Salmonella typhimurium (which has been safely administered to patients with metastatic melanoma and renal cell carcinoma). 25,30,31 Treatment of schwannoma models in mice is effective. Schwannomas are genetically stable, slow-growing, highly vascularized, and possess large hypoxic regions. These characteristics make schwannomas an ideal homing environment for bacteria and a potentially perfect target for bacterial cytotoxic and anti-angiogenic properties. Furthermore, the ability of bacteria to induce an immune response allows for the treatment of multiple distant lesions and the establishment of control mechanisms to prevent the recurrence of new schwannomas (a hallmark of these tumors) throughout the patient's life.

[0047] The methods described herein include approaches for treating benign nervous system tumors. In some embodiments, the tumor is a schwannoma. Schwannomas are composed of Schwann cells and form along peripheral, spinal, and cranial nerves. These tumors can cause pain, sensory / motor dysfunction, and death by compressing peripheral nerves, the spinal cord, and / or the brainstem. Multiple schwannomas in distal peripheral and intracranial nerves are hallmarks of neurofibromatosis 1 and 2 (NF1 and NF2) and schwannomatosis (three types of schwannoma). Schwannomas are benign tumors composed of tumor-dedifferentiated Schwann cells. Although usually non-malignant and slow-growing, these tumors can have devastating consequences for patients. They cause extreme pain and impair sensory / motor function, including hearing and vision. Schwannomas in NF2 are often associated with neurological deficits such as paresthesia, weakness, or hearing loss, and similar tumors in schwannomatosis frequently cause severe pain. Some schwannomas become very large, causing compression of adjacent organs or structures, and may lead to paralysis or death due to progressive spinal cord or brainstem compression. Schwannomas can occur sporadically without exhibiting any genetic characteristics of NF1, NF2, and schwannomatosis. Most vestibular schwannomas are sporadic, and therefore their incidence is very significant. Vestibular schwannomas usually occur as a single tumor rather than as multiple tumors throughout the body. In some implementations of any aspect, subjects requiring treatment for schwannomas can be subjects who have or have been diagnosed with a condition selected from the following groups: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), schwannomatosis, meningioma, schwannoma, schwannoma, vestibular schwannoma, sporadic schwannoma, neurofibrosarcoma, neurofibroma, neurofibromatosis (NF), malignant peripheral schwannoma, and combinations thereof. Subjects who can be treated using this method include mammals, such as humans and non-human veterinary subjects, such as cats, dogs, horses, goats, cattle, etc.

[0048] The current standard of care for patients with NF2 and schwannomas is surgical resection or radiosurgery to reduce the size of the symptomatic tumor. Unlike sporadic schwannomas (where only a single tumor is usually present, and surgery is generally an effective treatment strategy as long as the lesion is resectable), in schwannomas and NF2 with multiple tumors, resection often fails due to the difficulty in accessing many tumors and the risk of nerve damage, including major motor dysfunction, significant sensory loss (including deafness in the case of NF2 vestibular schwannomas), and neuropathic pain. Therefore, for most individuals, there is a significant incidence of schwannomas associated with both NF2 and schwannomas, and with current treatment options. This suffering and debilitating nature, along with the lack of treatment options, makes the treatment of schwannomas a major unmet medical need.

[0049] Typically, this method involves administering a therapeutically effective amount of attenuated Salmonella, optionally in combination with a checkpoint inhibitor, to a subject who requires or has been identified as requiring such treatment. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, and intratumoral (it) administration. In a preferred embodiment, the intratumoral route is used to maximize the bacterial dose and minimize potential dose-limiting toxicities (DLTs). Those skilled in the art will be able to identify the subject as having a benign neurological tumor. In some embodiments, the subject is a subject who has or has been diagnosed with a benign tumor or tumor-related condition selected from the group consisting of: neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), schwannoma, meningioma, schwannoma, vestibular schwannoma, sporadic schwannoma, neurofibroma, neurofibromatosis (NF), or any combination thereof. In some embodiments, the subject does not have a malignant solid tumor, e.g., does not have cancer. In some implementations, the subjects have a condition associated with an increased risk of benign nervous system tumors, such as neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), or schwannomatosis.

[0050] As used herein, the term "effective amount" refers to the amount of composition required to alleviate at least one or more symptoms of a disease or condition, and relates to an amount of pharmaceutical composition sufficient to provide the desired effect. Therefore, the term "therapeutic effective amount" refers to an amount of composition sufficient to provide a specific antitumor effect when administered to a typical subject. Effective amounts, as used herein, will also include, in various cases, an amount sufficient to delay the development of symptoms of a disease, alter the course of symptoms of a disease (e.g., but not limited to slowing the progression of symptoms of a disease), or reverse the symptoms of a disease. Therefore, specifying an exact "effective amount" is generally impractical. However, for any given situation, an appropriate "effective amount" can be determined by a person skilled in the art using only routine experiments. Administration of a therapeutically effective amount of the compounds described herein for the treatment of benign nervous system tumors may, for example, result in a reduction in tumor size, tumor number, tumor growth rate, or likelihood of recurrence after treatment with the methods described herein.

[0051] Therefore, this method includes administering attenuated Salmonella Typhimurium strains to inhibit tumor growth. As illustrated herein, to enhance therapeutic efficacy, in a preferred embodiment, this method may utilize intratumoral injection of the bacteria instead of intravenous delivery, which increases the bacterial concentration within the tumor and minimizes systemic toxicity. As illustrated herein, direct injection of attenuated Salmonella Typhimurium into a schwannoma has a vaccine-like effect, inducing an antitumor adaptive immune response.

[0052] Attenuated Salmonella Typhimurium

[0053] As used herein, the term “attenuated” refers to a strain that is reduced in virulence compared to the natural strain, thus becoming harmless or less virulent. Attenuation does not mean inactivation. In both strains, attenuation reduces the likelihood of pathogenicity, including septic shock. While current data primarily concern VNP20009 and ΔppGpp, other attenuated strains may also be used. Methods for generating attenuated Salmonella strains are known in the art and include directed or random mutagenesis followed by screening for reduced virulence. Directed mutations may be used, for example, in the aroA gene (aroA is part of the shikimic acid pathway that links glycolysis to the synthesis of aromatic amino acids; aroA-deficient Salmonella strains are described, for example, in Feiger et al., mBio, 2016, 7:e01220-16), the pull gene (purine synthesis defect), or the asd gene (aspartate-semialdehyde dehydrogenase defect required for cell wall synthesis). Attenuated strains of Salmonella have been disclosed in WO 2014 / 005683, WO 2016 / 202459, WO 2013 / 09189 and US 20200038496 (attenuated S. typhi Ty21a). The strains that can be used in this method include attenuated versions of the following strains: *Salmonella enterica* serovar typhimurium (“S. typhimurium”), *Salmonella montevideo*, *Salmonella enterica* serovar Typhi (“S. typhi”), *Salmonella enterica* serovar Paratyphi B (“S. paratyphi B”), *Salmonella enterica* serovar Paratyphi C (“S. paratyphi C”), *Salmonella enterica* serovar Hadar (“S. hadar”), and *Salmonella enterica* serovar... Salmonella enteriditis (“S. enteriditis”), Salmonella enterica serovar Kentucky (“S. kentucky”), Salmonella enterica serovar Infantis (“S. enteriditis”).Salmonella enterica serovar Pullorurn (“S. pullorum”), Salmonella enterica serovar Gallinarum (“S. gallinarum”), Salmonella enterica serovar Muenchen (“S. muenchen”), Salmonella enterica serovar Anaturn (“S. anatum”), Salmonella enterica serovar Dublin (“S. dublin”), Salmonella enterica serovar Derby (“S. derby”), and Salmonella enterica serovar Choleraesuis var. kunzendorf (“S. cholerae”). kunzendorf”) and Minnesota serotype entero Salmonella (Salmonella entericaserovar minnesota) (“S. minnesota”). See, for example, WO / 2008 / 039408 and US 20200023053; US20190153452; US 20170333490 and US 20180339032; Grant et al., PLoS Pathog. 2012 Dec; 8(12):e1003070; Tennant and Levine, Vaccine. 2015 Jun 19; 33(0 3):C36–C41.

[0054] In a preferred embodiment, the attenuated strain used in this method does not contain Clostridium novie (see, for example, WO2014160950). In a preferred embodiment, the attenuated strain used in this method does not contain a cleavage gene or cassette operatively linked to an intracellularly induced Salmonella promoter (see, for example, US 20170333490).

[0055] combination therapy

[0056] This approach may include administering attenuated Salmonella strains in combination with one or more other therapies. For example, this study showed that intratumoral VNP20009 in schwannomas of immunocompetent mice leads to tumor-adjuvant CD4 activation. + T cells and cytotoxic CD8 + Increased percentage of T cells and CD25 + A synchronous decrease in the percentage of Tregs. These changes in the tumor-infiltrating T cell population, along with the shift to M1 tumor-killing macrophages, indicate that *Salmonella typhimurium* induces an adaptive antitumor immune response. High PD-L1 expression reported in schwannomas suggests resistance to cell-mediated immunity within the tumor immune microenvironment. 49 Therefore, this study evaluated the efficacy of increased PD-1 immune checkpoint inhibition in controlling the growth of schwannomas associated with intratumoral Salmonella typhimurium (VNP20009) and its role in the development of host antitumor adaptive immunity. Data showed that this combination led to enhanced tumor regression in schwannomas injected with the bacteria, which is associated with CD4+. + Helper T cells and CD8 + Increased number of cytotoxic T cells and CD25 levels in both injected and uninjected tumors were observed. + A decrease in the number of regulatory T cells was associated (Figures 3 and 4). Although in contralateral schwannomas without bacterial injection ( Figure 3D E) and in re-excited schwannomas ( Figure 4D In E), compared with tumors injected with bacteria, the combination of VNP20009 and anti-PD-1 mAb resulted in the same enhancement of the T cell population (increased CD4+). + and CD8 + And reduced CD25 + However, there was no difference in growth inhibition between VNP20009 / anti-PD-1 mAb and VNP20009 alone. On the other hand, the effects of intratumoral VNP20009 and the VNP20009 / anti-PD-1 mAb combination on the control of schwannoma growth were observed in re-excited tumors. Figure 4D It appears to be more pronounced than in primary tumors where bacteria were injected. Figure 4B The differences in the biological composition of the schwannomas that were injected with bacteria, the uninjected contralateral schwannomas, and the re-inflamed schwannomas that were larger remain to be elucidated.

[0057] Therefore, this method may include administering (together or separately) a combination of bacteria and a checkpoint inhibitor, such as an inhibitor of PD-1 signaling, for example, an antibody that binds to PD-1, CD40 or PD-L1, or an inhibitor of Tim3 or Lag3, for example, an antibody that binds to Tim3 or Lag3, or an antibody that binds to CTLA-4.

[0058] Exemplary anti-PD-1 antibodies that can be used in the methods described herein include those that bind to human PD-1; an exemplary PD-1 protein sequence is provided with NCBI accession number NP_005009.2. Exemplary antibodies are described in US8008449, US9073994, and US20110271358, including PF-06801591, AMP-224, BGB-A317, BI 754091, JS001, MEDI0680, PDR001, REGN2810, SHR-1210, TSR-042, pembrolizumab, nivolumab, avelumab, pidilizumab, and atezolizumab.

[0059] Exemplary anti-CD40 antibodies that can be used in the methods described herein include those that bind to human CD40; exemplary CD40 protein precursor sequences are provided with NCBI accession numbers NP_001241.1, NP_690593.1, NP_001309351.1, NP_001309350.1 and NP_001289682.1. Exemplary antibodies include those described in WO2002 / 088186, WO2007 / 124299, WO2011 / 123489, WO2012 / 149356, WO2012 / 111762, WO2014 / 070934, US20130011405, US20070148163, US20040120948, US20030165499, and US8591900, including dacetuzumab, lucarumumab, bleselumab, teneliximab, ADC-1013, CP-870,893, and Chi Lob. 7 / 4, HCD122, SGN-4, SEA-CD40, BMS-986004, and APX005M. In some implementations, the anti-CD40 antibody is a CD40 agonist rather than a CD40 antagonist.

[0060] Exemplary CTLA-4 antibodies that may be used in the methods described herein include those that bind to human CTLA-4; an exemplary CTLA-4 protein sequence is provided with NCBI accession number NP_005205.2. Exemplary antibodies include those described in Tarhini and Iqbal, Onco Targets Ther. 3:15-25 (2010), Storz, MAbs. 2016 Jan; 8(1):10–26, US2009025274, US7605238, US6984720, EP1212422, US5811097, US5855887, US6051227, US6682736, EP1141028, and US7741345; and include ipilimumab, tremelimumab, and EPR1476.

[0061] Exemplary anti-PD-L1 antibodies that can be used in the methods described herein include those that bind to human PD-L1; exemplary PD-L1 protein sequences are provided with NCBI accession numbers NP_001254635.1, NP_001300958.1, and NP_054862.1. Exemplary antibodies are described in US20170058033, WO2016 / 061142A1, WO2016 / 007235A1, WO2014 / 195852A1 and WO2013 / 079174A1, including BMS-936559 (MDX-1105), FAZ053, KN035, Atezolizumab (Tecentriq, MPDL3280A), Avelumab (Bavencio), and Durvalumab (Imfinzi, MEDI-4736).

[0062] Exemplary anti-Tim3 (also known as hepatitis A virus cell receptor 2 or HAVCR2) antibodies that can be used in the methods described herein include those that bind to human Tim3; an exemplary Tim3 sequence is provided with NCBI accession number NP_116171.3. Exemplary antibodies are described in WO2016071448; US8552156; and US PGPub.Nos.20180298097, 20180251549, 20180230431, 20180072804, 20180016336, 20170313783, 20170114135, 20160257758, 20160257749, 20150086574, and 20130022623, and include LY3321367, DCB-8, MBG453, and TSR-022.

[0063] Exemplary anti-Lag3 antibodies that may be used in the methods described herein include those that bind to human Lag3; an exemplary Lag3 sequence is provided with NCBI accession number NP_002277.4. Exemplary antibodies are described in Andrews et al., Immunol Rev. 2017 Mar; 276(1):80-96; Antoni et al., Am Soc Clin Oncol Educ Book. 2016; 35:e450-8; US PGPub.Nos. 20180326054, 20180251767, 20180230431, 20170334995, 20170290914, 20170101472, 20170022273, 20160303124, and include BMS-986016.

[0064] This method may also include administering a combination of bacteria and angiogenesis inhibitors (together or separately). Many angiogenesis inhibitors are known, including those that target vascular endothelial growth factor (VEGF), its receptor (VEGFR), or other molecules involved in angiogenesis. Specific examples include axitinib (INLYTA), bevacizumab (AVASTIN), cabozantinib (COMETRIQ), everolimus (AFINITOR), lenalidomide (REVLIMID), lenvatinib mesylate (LENVIMA), pazopanib (VOTRIENT), ramucirumab (CYRAMZA), regorafenib (STIVARGA), sorafenib (NEXAVAR), sunitinib (SUTENT), thalidomide (SYNOVIR, THALOMID), vandetanib (CAPRELSA), or aflibercept (ZALTRAP). See, for example, Zhang et al., Exp Neurol. 2018 Jan; 299(Pt B):326-333; de Vries et al., Otol Neurotol. 2015 Aug; 36(7):1128-36; Lim et al., Cancer Treat Rev. 2014 Aug; 40(7):857-61; Blakeley, Curr Opin Otolaryngol Head Neck Surg. 2012 Oct; 20(5):372-9; Goel et al., Cold Spring Harb Perspect Med. 2012 Mar; 2(3):a006486.

[0065] Optionally or additionally, this method may be used in combination with surgical resection. For example, in some embodiments of any aspect, attenuated Salmonella strains as described herein may be administered before, simultaneously with, or after surgical resection or partial resection of vegetations or tumors such as schwannomas. Various treatment methods of the present invention may further include treating the subject with surgery, radiation therapy, or chemotherapy, or combinations thereof.

[0066] Pharmaceutical Compositions and Administration

[0067] The methods described herein involve using pharmaceutical compositions containing attenuated Salmonella as the active ingredient. Pharmaceutical compositions typically include pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" includes saline, solvents, and dispersion media compatible with drug administration. Complementary active compounds may also be introduced into the composition, such as checkpoint inhibitors and / or angiogenesis inhibitors, for example, those known in the art and / or discussed herein.

[0068] Pharmaceutical compositions are typically formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, subcutaneous administration, and intratumoral administration.

[0069] Example

[0070] The invention is further described in the following embodiments, which do not limit the scope of the invention as described in the claims.

[0071] Materials and methods

[0072] The following materials and methods are used in the embodiments listed below.

[0073] Cell culture

[0074] The HEI-193 human schwannoma cell line (from DJLim, House Ear Institute, Los Angeles, CA) was established from schwannomas in patients with NF2, immortalized with the human papillomavirus E6 / E7 gene, and grown as described. 68,69 Mouse 08031-9 schwannoma cells (from Dr. Marco Giovannini, Univ. of California, Las Angeles, CA) grew as described. 50 Cell lines were infected with lentiviruses encoding Fluc (firefly luciferase) and mCherry, respectively, for bioluminescence imaging and IHC. 70 Human MPNST (STS.26T) cells were kindly provided by Dr. David Largaespada (Masonic Cancer Center, University of Minnesota) and grew as described. 56Human NF-1-associated MPNST (S462TY) cells were kindly provided by Dr. Timothy P. Crepe (Children's Cancer Center, National Children's Hospital). Human Ben-Men-1 and CH-157 cells were kindly provided by Dr. Long-Sheng Chang (Children's Cancer Center, National Children's Hospital) and Dr. G. Yancey Gillespie (University of Alabama at Birmingham), respectively. For the differentiation of human macrophages, phorbol-12-myristate 13-acetate (PMA) (Sigma-Aldrich, USA) was added to human monocytes at a final concentration of 100 nM. After 24 hours, the PMA-supplemented medium was removed, the cells were washed with PBS, and then incubated for another 24 hours in fresh PMA-free medium to obtain macrophage phenotypic characteristics. 71 Mouse RAW macrophages (from ATCC, USA) were obtained. The macrophages were cultured in RPMI medium according to the manufacturer's instructions. All cell lines were confirmed to be free of contamination, including mycoplasma, prior to experimental use.

[0075] Bacterial culture

[0076] The attenuated Salmonella enterica serotype VNP20009 (with modified lipid A (msbB–), purine auxotroph mutant (purI–)) (from ATCC, USA, cat#14028) and strain ΔppGpp (with defective ppGpp synthesis (RelA::cat, SpoT::kan)) were kindly provided by Dr. Karsten Tedin, Institute of Microbiology and Epidemiology, Center for Infectious Diseases, Berlin, Germany. As previously documented. 31,47 Bacterial cells were cultured overnight at 37°C under aerobic conditions at 300 rpm in low-sodium Luria-Bertani (LB) broth (Difco Laboratories, USA). In short, cells were allowed to grow to late logarithmic phase (OD 600 nm = 0.8) and collected by centrifugation at 5000 rpm for 10 min. Cells were washed twice with sterile 1× phosphate-buffered saline (PBS) before injection into tumors or infection of cultured cells.

[0077] animal

[0078] All animal experiments were approved and conducted under the supervision of the Institutional Animal Care and Use Committee (IACUC Protocol No. 2014N000211) of Massachusetts General Hospital (MGH, Boston, MA). Five- to seven-week-old male mice (nu / nu and FVB / N, Charles River Laboratories) were kept in a 12:12 light-dark cycle with free access to food, water, and daily health checks by staff / veterinarians at the Center for Comparative Medicine of MGH.

[0079] Animal models and intratumoral bacterial injection

[0080] As recorded 72 Sciatic nerve schwannomas were generated by directly injecting HEI-193FC human or 08031-8FC mouse schwannomas into the left sciatic nerve of mice anesthetized with isoflurane. HEI-193FC or 08031-9FC cells were digested with trypsin and washed with cold PBS. Using a glass micropipette and a pneumatic microsyringe (IM-300; Narishige, Tokyo, Japan), 30,000 cells (or 10,000 for 08031-9FC) in 0.5 μl PBS were injected into the sciatic nerve of athymic nude mice (nu / nu, 5-7 week old males; National Cancer Institute [NCI]) or syngeneic FVB / N mice (5-7 week old males; Charles River Laboratory). Two weeks after HEI-193 tumor cell implantation or one week after 08031-9 tumor cell implantation, 10 tumor cells were injected into the sciatic nerve of mice in 2 μl PBS. 4 CFU-attenuated Salmonella typhimurium (VNP20009 or ΔppGpp) [9, 13], targeting the site of implanted tumor cells on the sciatic nerve. As documented. 72Tumor growth was monitored in vivo using bioluminescence imaging, once weekly for HEI-193 and twice weekly for 08031-9. Briefly, mice were intraperitoneally injected with the Fluc substrate d-luciferin, and the signal was acquired using a high-performance IVIS Spectrum (Caliper Life Sciences, Hopkinton, MA) 10 min later. For immunocompetent subcutaneous models, 08031-9 was resuspended in DMEM and mixed with Matrigel (BD bioscience) (1:1), then subcutaneously (sc) transplanted into FVB / N syngeneic mice. For NF-1 or meningioma xenograft models, as described... 56 Human S462TY cells, STS.26T cells, Ben-Men-1 cells, or CH-157 cells were mixed with matrix gel (1:1) and subcutaneously implanted into nu / nu mice. Tumor volume was estimated using the formula (W×L×L×π / 6), where width (W) and length (L) are the two largest diameters. 50 As previously recorded. 51 When the tumor reaches 150mm 3 Treatment began with intraperitoneal (ip) PD-1 mAb (250 μg / injection, Bio X Cell, USA) and was repeated every three days. VNP20009 was injected intratumorally using an insulin injector (10 μg / injection). 4 CFU / 100μl PBS). Mice in the control group were treated with PBS solvent or allotype antibody according to the same protocol.

[0081] Histological analysis and immunohistochemical analysis

[0082] Finally, the animals were anesthetized with isoflurane (3%) and euthanized by decapitation. As described in the records... 73Tumor tissue was removed and flash-frozen for hematoxylin and eosin (H&E) and immunohistochemical staining. The tumor was stored in an OCT block at -80°C. Sections were stained with H&E according to standard protocol. Proliferation marker staining was performed using an antibody against Ki67 (Abcam, Cambridge, MA). Antibodies against CD45, CD68, and CD31 were used for staining leukocytes, macrophages, and angiogenesis, respectively. All antibodies were purchased from (Abcam, Cambridge, MA). Briefly, sections were dried overnight at room temperature (RT). They were fixed in pre-chilled acetone at 4°C for 10 min, dried, and then stained immediately. Sections were washed in PBS, blocked with a serum-free protein block (Dako, Carpinteria, CA), and peroxidase was quenched in a dual endogenous enzyme block (Dako). Sections were washed in PBS, incubated with primary antibody at room temperature for 1 hour, washed again in PBS, and incubated with horseradish peroxidase-conjugated secondary antibody at room temperature (RT) for 30 min. Sections were washed in PBS and incubated with DAB solution (Dako). Counterstaining was performed by immersing sections in ethanol and xylene, then mounted in a Cytoseal (Richard Allan Scientific, San Diego, CA) and covered with coverslips for microscopic observation. In vivo apoptosis staining was assessed using the TACS 2TdT-DAB in situ apoptosis detection kit (Trevigen, Gaithersburg, MD). 15 μm sections of freshly frozen nerves fixed with 3.7% formaldehyde (cryostat) after being placed on slides were stained with diaminobenzidine (DAB) and observed under an optical microscope according to the manufacturer's instructions. The antibodies used are provided in Table 1.

[0083] Table 1. Antibodies used in the study

[0084]

[0085] Quantitative real-time PCR (qRT-PCR) was used to measure cytokine RNA in tumors.

[0086] Tumor tissue was removed on day 3 after bacterial injection, and RNA was extracted using Trizol. SuperScript was used with the ezDNase enzyme (Invitrogen). TM IV VILO TMTotal RNA was transcribed into cDNA. Samples were then incubated at 37°C for 10 min to digest the DNA, followed by incubation at 25°C for 10 min, 50°C, and 85°C for 5 min each on a ProFlex PCR system (Applied Biosystems, USA). For qPCR reactions, 20 ng cDNA / well was used as input to determine target gene expression using Taq-man probes. qPCR was performed on an Mx3000P qPCR system (Agilent Technologies, USA) using standard cycling conditions. Melting curve analysis was performed using MxPro qPCR software to validate primer efficiency on each plate and exclude any non-specific amplification. The difference in cycling thresholds (Ct) (ΔCt) between the target gene and the reference gene at 18S was converted to relative expression using the 2-ΔΔCT method, and fold change was calculated by comparing samples. All reactions were performed in triplicate.

[0087] ELISA was used to measure cytokine proteins in tumors.

[0088] Tumor tissue was excised on day 3 post-bacterial injection and homogenized in NP40 lysis buffer containing protease inhibitors. The supernatant was collected by centrifugation at 13,000 rpm for 10 min. Cytokine levels were measured using the following individual Quantikine ELISA kits (R&Dsystems, Minneapolis) for humans and mice, following the manufacturer's instructions: interferon-gamma (IFN-γ) (BD bioscience), TNF-α (BD bioscience), IL-1β / IL-1F2 (BD bioscience), and IL-18 (BD bioscience). Substrate color reactions were measured at 450 nm using a microplate reader (SpectraMax, Molecular Devices) with the calibration wavelength set at 540 nm or 570 nm, and the results were then quantified using a standard curve.

[0089] In vitro invasiveness test

[0090] Macrophages (human THP-1 and mouse RAW 264.7 macrophages) and schwannomas (human HEI-193 and mouse 08031-9) were grown to a density of 10 cells per well in 24-well tissue culture plates. 4Cells were washed with warm PBS and replenished with antibiotic-free 10% FBS medium. Similarly, bacterial cells were grown to late logarithmic phase as previously described and diluted in cell culture medium to represent a 50:1 bacteria / cell multiplicity of infection (MOI). The culture medium containing the bacteria was added to the cultured macrophages and schwannomas and incubated at 37°C for 60 minutes. To determine the invasiveness of the bacterial strain, the cultured cells were washed with PBS and incubated for 30 minutes in medium containing gentamicin sulfate (50 μg / mL) to kill any extracellular bacteria attached to the cell surface. The cells were then washed five times with 1–2 mL of PBS, followed by the addition of 0.2 mL of 0.1% Triton X-100 for 10 minutes to induce cell lysis and detachment of attached bacteria. LB broth (0.8 mL) was then added, and the samples were vigorously mixed to prepare a homogeneous suspension for serial dilution. Prepare a 10-fold dilution and plate it on LB agar medium and incubate it overnight at 37°C to count colony forming units (CFU).

[0091] Flow cytometry

[0092] Tissues were collected from mice (n=3 / group), and cells were lysis buffered in a water bath at 37°C for 1 hour using freshly prepared lysis buffer (125 U / mL type XI collagenase, 60 U / mL type I1 hyaluronidase, 60 U / mL DNase 1, and 450 U / mL type I collagenase in PBS containing 20 mM Heps, Sigma-Aldrich), with gentle tapping every 10 minutes for proper homogenization and cell dissociation. The cell suspension was then passed through a pre-wetted 70 μm cell filter (BD-Falcon). Cell quantification was performed by mixing 10 μL of the suspension with 10 μL of trypan blue and loading it onto a hemocytometer. The cell suspension was centrifuged at 2000 rpm for 10 minutes at 4°C to remove the lysis buffer, washed, and resuspended in 1×PBS to maintain a 10-day lysis buffer level. 6Cells / 100 μL. Cells were incubated for 15 min in 2 μL of FC blocking agent (BD Biosciences) at room temperature. Cells were washed with PBS and then incubated in the dark for 1 h with fluorescently labeled antibodies against cell surface markers or different immunomarkers, followed by a washing step with PBS and permeabilization overnight with 2% paraformaldehyde (PFA solution). Antibodies against the following mouse immunomarkers were used for surface staining: CD45, F4 / 80, CD206, CD86, LY6G, NK1.1, NKp46, CD11b, CD11c, CD4, CD8, CD3, CD25. FACS and analysis were performed using FACSDiva software (BD Biosciences) and FlowJo software, and using FACSAria and LSR Tortessa. The antibodies used are listed in Supplementary Table 1.

[0093] Data Analysis

[0094] All data are expressed as group mean ± standard error of the mean (SEM). Data were analyzed using GraphPad Prism and Microsoft Excel. As documented... 74 Repeated measures ANOVA was used to compare tumor volume and / or signal. One-way ANOVA was used to analyze cytokine expression and flow cytometry data. P < 0.05 was considered statistically significant.

[0095] Example 1. Intratumoral attenuated Salmonella typhimurium injection inhibits tumor growth in xenograft human schwannoma and allogeneic mouse schwannoma models.

[0096] The inventors evaluated whether intratumoral (it) injection of Salmonella Typhimurium could control the growth of human (HEI-193 cell line) schwannomas and mouse (08031-9 cell line) schwannomas occurring in the sciatic nerve of null / null immunocompromised mice and FVB / N immunocompetent mice, respectively. Two different Salmonella Typhimurium strains, VNP20009 and ΔppGpp, were evaluated. Both strains are attenuated mutant forms of wild-type bacteria and were used in preclinical studies. 19,32,33 It shows higher tumor tropism and improved safety, and VNP20009 is also being investigated in clinical trials. 25,34 It showed higher tumor tropism and improved safety.

[0097] Tumor burden was assessed by in vivo bioluminescence imaging of firefly luciferase (Fluc) expressed in HEI-193FC (human NF2 schwannoma) cells and 08031-9FC (mouse NF2-deficient schwannoma) cells. Tumor signaling was stabilized (approximately 2 weeks or 1 week after tumor implantation into HEI-193FC cells or 08031-9FC cells, respectively). Figure 1A &B), under direct observation, attenuated Salmonella typhimurium (VNP20009 or ΔppGpp) or PBS (control) was injected into the tumor-bearing sciatic nerve (n = 8 mice / group). Tumor growth was tracked for 5 weeks in nude mice carrying human NF2 schwannomas and for 2 weeks in immunocompetent FVB / N mice carrying mouse NF2 schwannomas. In the xenograft schwannoma model, the study was terminated 7 weeks after tumor cell implantation (at which point most bacterially injected tumors showed no bioluminescent signal). The study in the intrasciatic allogeneic transplantation model was terminated based on the development of motor dysfunction in the tumor-bearing hindlimb of the control mice (assessed by the inventors' team and animal care technicians who were unaware of the study group). Two additional replicates of this study were performed in the xenograft model and one replicate in the allogeneic transplantation model; (n = 8 mice / group for all studies). Figure 7A &B).

[0098] In the xenograft human NF2 schwannoma model, all three replicates (p<0.01) were performed. Figure 1A ; Figure 7A () and two replicates in an allogeneic mouse schwannoma model (p<0.05, Figure 1B ; Figure 7B In this study, intratumoral injection of attenuated Salmonella Typhimurium strain VNP20009 resulted in a reduction in bioluminescent tumor signal compared to the PBS control. As early as one week after VNP20009 injection, growth curves in both bacterial-injected and PBS-injected mice began to diverge in both tumor models. At week 2 post-injection, in 5 out of 8 mice, bacterial treatment reduced the HEI-193FC tumor signal to undetectable levels. Figure 1A In three replicates of this experiment, 75% (18 / 24 mice) of the animals injected with VNP20009 showed no detectable tumor signal at the end of the experiment. Figure 1A , Figure 7A ).

[0099] Although the inventors did not observe complete regression of tumor signal after bacterial injection in an allogeneic mouse schwannoma model, tumor growth control persisted until sacrifice. At this point, the bioluminescent signal in mice injected with VNP20009 was approximately 8-fold lower than that in the PBS control. Figure 1B , Figure 7B ).

[0100] The inventors then tested whether the ΔppGpp strain of *Salmonella typhimurium* possessed similar therapeutic efficacy to VNP20009. Although the effects of VNP20009 and ΔppGpp on tumor signaling were indistinguishable in the xenograft model, in the allogeneic mouse schwannoma model, VNP20009, rather than the ΔppGpp strain, controlled tumor growth (p<0.05 for VNP20009 against PBS). Figure 1B Furthermore, there was a significant difference between the two strains (p < 0.05 for VNP20009 versus ΔppGpp). Figure 1B ).

[0101] Histological analysis of tumor-bearing nerves (n = 3 mice / group) collected at the end of the experiment (i.e., 5 weeks after bacterial injection in the xenograft human schwannoma model and 2 weeks after bacterial injection in the allogeneic mouse schwannoma model) showed that tumors injected with both VNP20009 and ΔppGpp had a large number of apoptotic bodies compared with tumors injected with PBS. Figure 1C &D). Quantitative analysis of tissues from xenograft models showed that, compared with tumors injected with PBS (4±1), both VNP20009-injected schwannomas (570±77; p<0.0005) and ΔppGpp-injected schwannomas (230±68; p<0.005) had more apoptotic bodies (P<0.005). Figure 1C A comparison of apoptotic bodies induced by the two bacterial strains showed that, in HEI-193 schwannomas, tumors treated with VNP20009 had a greater number of apoptotic cells compared to tumors treated with ΔppGpp. (p<0.01, Figure 1C In xenograft human NF-2 schwannomas, VNP2009 injection resulted in approximately three times more apoptotic cells than ΔppGpp injection. Consistent intergroup differences were observed using an allogeneic mouse schwannoma model; tumors injected with VNP2009 contained more apoptotic cells (340±63) compared to tumors injected with ΔppGpp (110±27, p<0.01) and tumors injected with PBS (6±0.3, p<0.001). Tumors injected with ΔppGpp also had a significantly higher number of apoptotic cells compared to the PBS control (p<0.01). Figure 1D In this model, intratumoral VNP20009 injection resulted in approximately three times more apoptotic cells than ΔppGpp injection (p<0.01). Figure 1D ).

[0102] Example 2. Intratumoral injection of attenuated Salmonella typhimurium induced increased pro-immunogenic cytokines and altered immune cell infiltration in an allogeneic mouse schwannoma model.

[0103] One of the most exciting properties of BCT is its ability to induce anti-tumor adaptive immunity. 35-37 Based on this discovery, the inventors hypothesize that infecting schwannomas with attenuated Salmonella typhimurium would have a vaccine-like effect, inducing adaptive anti-tumor immunity in the host. Considering that affected individuals typically have multiple tumors, develop new tumors throughout their lives, have tumors in sites that are not surgically resectable without a significant risk of major neuronal damage, and that complete resection is often infeasible and, as mentioned, tumors will occur throughout life requiring multiple surgeries, immunotherapy for schwannomas would be particularly valuable.

[0104] Although intratumor injection of bacterial bacteria into the xenograft and allogeneic schwannoma models of the present invention resulted in apoptotic cell death (Figure 1), the inventors wanted to investigate whether there was also evidence of pyroptosis and / or immunogenic cell death. Therefore, the inventors tested whether intratumor injection of VNP20009 and ΔppGpp into human HEI-193 schwannomas and mouse 08031-9 schwannomas grown in the sciatic nerve of nude mice and immunocompetent mice could induce a wide range of innate and adaptive immune responses in the host. The inventors analyzed the lymphocyte common antigen marker CD45... 38 And CD68, a marker for monocytes and tissue macrophages 39 To evaluate intratumoral immune cell infiltration, immunocytochemical staining of tumors was performed (n = 3 tumors / treatment / model) collected 5 weeks after bacterial injection in a xenograft human NF2 model and 2 weeks after bacterial injection in an allogeneic mouse schwannoma model. The timing of sacrifice was chosen for the aforementioned reasons—namely, to ensure that tumor signal regression occurred in most mice (xenograft models) or just before significant morbidity was observed in control mice (allogeneic transplant models).

[0105] In xenograft models, histological analysis of tumor-bearing nerves showed CD45 levels compared to tumors injected with PBS that showed no signs of any cell type. + White blood cells and CD68 + Extensive tumor infiltration by macrophages ( Figure 8 Similar analyses of intraisical allogeneic rat schwannomas showed that intratumoral injection of VNP20009 or ΔppGpp resulted in increased CD45 levels in tumors compared to those injected with PBS. + White blood cells and CD68 + Macrophage infiltration ( Figure 2ACD45 in the tumor microenvironment of an allogeneic transplantation model + Cells and CD68 + Quantitative analysis of the cells showed that injection of this bacteria resulted in an increase in tumor-infiltrating leukocytes and macrophages compared to a control group injected with PBS. Figure 2B ).

[0106] As these tumors developed in immunocompetent host mice, using only an allogeneic transplantation model, the inventors then focused on characterizing the cytotoxic effect of *Salmonella typhimurium* on schwannomas to investigate the presence of immunogenic cellular responses and signs of immunogenic cell death. Macrophages could be classified into M1 tumoricidal and M2 tumorigenic types and appeared to promote (M1) tumorigenicity, respectively. 40 Or inhibit (M2) 41 Host-mediated antitumor adaptive immunity is crucial, and a key determinant of this immunity is the balance between M1 and M2 macrophages. Human schwannomas have been reported to consist of macrophages comprising up to 50% of their cell count. 42 Furthermore, a higher macrophage count is associated with a higher tumor growth rate. 43 The inventors collected CD45 samples from mouse schwannomas transplanted intraisally 3 and 7 days after intratumoral injection of attenuated Salmonella typhimurium. + F4 / 80 + CD86 in cells + (For M1 type, oncolytic) and CD206 + (For M2 type, tumorigenicity) expression was evaluated by flow cytometry to assess the macrophage population. The inventors found that, compared to PBS injection, intratumoral injection of both VNP20009 and ΔppGpp resulted in a macrophage balance shift towards the M1 type 3 days after bacterial injection, increasing the M1 / M2 macrophage ratio (M1 / M2) (p<0.05). Figure 2C Seven days after intratumoral bacterial injection, compared with PBS-treated tumors, the M1 / M2 ratio in tumors injected with VNP20009 further shifted towards M1 (p<0.01). Figure 2C In tumors injected with ΔppGpp, the M1 / M2 ratio decreased compared to the 3-day time point and was no longer different from that of PBS. Figure 2C Interestingly, there was a systemic effect of intratumoral attenuated Salmonella typhimurium injection on macrophage numbers. Three days after intratumoral bacterial injection, compared with PBS (15.4%), splenic macrophages (CD45) showed a significantly higher number of macrophages (CD45). + F4 / 80 + The incidence was increased in the VNP20009 group (38.8%, p<0.01) and the ΔppGpp group (32.4%, p<0.01). Figure 9 ).

[0107] Given the observed increase in tumor-infiltrating lymphocytes and the conversion to M1 macrophages in allogeneic schwannomas injected with bacteria, the inventors investigated whether intratumoral T cell composition was altered by intratumoral injection of attenuated Salmonella Typhimurium. Multicolor flow cytometry was used to assess tumor-infiltrating helper T cells (CD3 / CD4), cytotoxic T cells (CD3 / CD8), and regulatory T cells (Treg, CD4 / CD25) 7 days after intratumoral bacterial injection (n=3 / group). Although the inventors did not observe an effect of intratumoral bacterial injection on CD4+ helper T cells, intratumoral injection of VNP20009 or ΔppGpp increased the percentage of CD8+ cytotoxic T cells compared to PBS injection (7.56%, 7.56%, and 2.79%, respectively). Figure 2D Furthermore, intratumoral injection of VNP20009 or ΔppGpp, compared to PBS, reduced tumor-invasive CD25 levels. + The number of Tregs decreased (by 4.15%, 3.24%, and 8.32%, respectively). Figure 2D In all cases, these percentages represent CD45. + The proportion of cells.

[0108] The inventors then investigated the effects of intratumoral VNP20009 and ΔppGpp injections on two key immunostimulatory cytokines: tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) (which are associated with ICD and are known to regulate the survival, proliferation, and differentiation of both immune cells and tumor cells). 44,45 The inventors hypothesize that bacterial infection of schwannomas induces the production of these cytokines, partly due to the intratumoral attenuated Salmonella typhimurium leading to the transformation into M1-type macrophages. Figure 2C Furthermore, Salmonella typhimurium is involved in the processing and maturation of two pro-inflammatory cytokines (IL-1β and IL-18, known to have anti-tumor activity) and inflammasomes (including NLRP3 and NLRC4). 46 Known inducers 19,47,48 On day 3 after intratumoral bacterial injection, when the changes in the M1 / M2 ratio mediated by VNP20009 and ΔppGpp were significant ( Figure 2C The inventors observed an increase in multiple cytokines in intrasciatic allogeneic schwannomas. mRNA and proteins were extracted from the tumors, and the cytokine profile was assessed using RT-PCR and ELISA (N=3 / group). Compared to controls, the transcriptional levels of pro-inflammatory cytokines TNF-α, IFN-γ, IL-1β, and IL-18 were upregulated in tumors injected with VNP20009 and ΔppGpp Salmonella typhimurium. Figure 2ENote that tumors injected with VNP20009 showed higher levels of TNF-α and IFN-γ mRNA expression compared to ΔppGpp (p<0.01, p<0.0001, respectively). As observed by the inventors at the transcriptional level, compared to the PBS control, the protein levels of TNF-α, IFN-γ, IL-1β, and IL-18 were elevated in tumors injected with both VNP20009 and ΔppGpp. Figure 2F Furthermore, intratumoral VNP20009 injection, compared to ΔppGpp, resulted in higher levels of IL-18, IFN-γ, and TNF-α proteins in the injected tumor (p<0.05, p<0.01, and p<0.05, respectively). Figure 2F Compared with ΔppGpp or PBS, VNP20009 treatment also resulted in a greater increase in NLRC4 and NLRP3 mRNA (p<0.01). Figure 2E ).

[0109] Example 3. Intratumoral injection of Salmonella Typhimurium VNP20009 to control the growth of schwannomas in allogeneic mice with injected bacteria and in contralateral allogeneic mice without injection.

[0110] It has been confirmed that some schwannomas contain CD4 cells that express PD-1. + T cells and CD8 + T cells indicate that the anti-tumor immune function of these cells is impaired. 49 The inventors have discovered that intratumoral VNP20009 injection in allogeneic transplanted schwannomas is related to CD8. + Increased number of cytotoxic T cells and CD25 + A decrease in the number of Tregs is associated with the activation of an adaptive immune response. Therefore, the inventors evaluated that combining systemic anti-PD-1 monoclonal antibody (mAb) with intratumoral VNP20009 injection enhances the bacterial-induced host anti-tumor adaptive immune response. FVB / N mice were subcutaneously implanted bilaterally with 08031-9 mouse schwannoma cells and divided into four groups ( Figure 3A Experimental design (shown): i) Intratumoral injection of VNP20009 (left tumor), ii) Intraperitoneal injection of anti-PD-1-mAb P, iii) Intratumoral injection of VNP20009 and intraperitoneal injection of anti-PD-1-mAb, and iv) Intratumoral injection of PBS (left tumor). Subcutaneous implantation was used instead of intraisical implantation because the former allows for longer survival, thus providing a greater chance for adaptive immune response. Once the average tumor size reached approximately 150 mm... 3 50 (Day 11 post-implantation), only VNP20009 (10 in 100 μl) was administered. 4CFU or PBS was injected directly into the tumor on the left side. Simultaneously, anti-PD-1 mAb (250 μg / injection) was administered intraperitoneally on days 10, 13, 16, and 19 post-implantation. 51 The inventors observed that, compared with intratumoral PBS injection, monotherapy with VNP20009 or anti-PD-1 mAb inhibited the growth of both tumors. Figure 3B Furthermore, compared to PD-1-mAb, intratumoral VNP2009 resulted in stronger growth control in uninjected tumors (p<0.05). Figure 3B (D). The combination of intratumoral VNP20009 and anti-PD-1 mAb resulted in 1) enhanced tumor growth control of the injected bacteria compared to VNP20009 or anti-PD-1 mAb alone (p<0.05). Figure 3B ), and 2) showed enhanced tumor growth control in untreated contralateral tumors compared to anti-PD-1 mAb treatment (p<0.05), but no difference compared to VNP20009-treated mice ( Figure 3D ).

[0111] These effects on schwannoma growth suggest that intratumoral VNP20009 generates an adaptive immune response capable of controlling tumor growth, and that this effect can be enhanced by immune checkpoint inhibition. In light of this, the inventors analyzed T cell subsets in these tumors using flow cytometry, specifically helper (CD3 / CD4) T cells, cytotoxic (CD3 / CD8) T cells, and regulatory (CD4 / CD25) T cells. Figure 3C In the tumor on the left side ( Figure 3B C) Intratumoral VNP20009 and systemic anti-PD-1 mAb led to an increase in CD8 compared to PBS injection (1.01%). + 1) Increased cytotoxic T cells (11.8% and 10.8%, respectively), 2) increased CD4+ helper T cells compared to PBS injection (0.77%) (VNP20009 (3.43%); anti-PD-1 mAb (4.21%)), and 3) decreased regulatory T cells compared to PBS (31.2%) (VNP20009 (15.5%); anti-PD-1 mAb (19.3%)). The combination of VNP20009 and anti-PD-1 mAb resulted in increased CD8+ cells compared to either single therapy or PBS. + (24.9%) T cells and CD4 + (29.6%) Additive increase in T cells ( Figure 3CCompared with each single therapy (VNP20009 (15.5%); anti-PD-1 mAb (19.3%)) and PBS (31.2%), there was also an additive effect of the combination of bacterial and immune checkpoint inhibition on Treg suppression (7.59%).

[0112] To further evaluate whether these actions induced a systemic host antitumor immune response, the inventors analyzed the same T cell population in right-sided tumors that had never received bacterial injection. Intratumoral VNP20009 in combination with systemic anti-PD-1 mAb (in the left-sided tumor) resulted in increased activity against invasive CD4+ cells in uninjected tumors compared to VNP20009 alone (4.55%) or anti-PD-1 mAb alone (3.26%). + Synergistic effect of helper T cells (23.4%); no difference between monotherapy and PBS (2.24%). Figure 3E Compared with anti-PD-1 mAb (6.05%) and PBS treatment (4.92%), the VNP20009 / anti-PD-1 mAb combination (47.5%) or VNP20009 (41.9%) reduced CD8+ in the right-sided tumor. + The percentage of cytotoxic T cells increased, and the VNP20009 / anti-PD-1 mAb combination was greater than VNP20009 alone. Figure 3E Finally, as Figure 3E As shown, compared with intratumoral PBS injection (for the contralateral left-sided tumor), all treatment regimens reduced tumor-invasive CD25 levels. + The percentage reductions in Tregs were as follows: VNP20009 / anti-PD-1 mAb (14.2%), VNP20009 (22.2%), anti-PD-1 mAb (31.4%), and PBS (50.5%). Note that the effect on Treg reduction was greatest in VNP20009 / anti-PD-1 mAb mice, and VNP20009 had a greater effect than anti-PD-1 mAb.

[0113] Example 4. Intratumoral injection of Salmonella typhimurium (VNP20009) into primary allogeneic transplanted mouse schwannomas inhibited the growth of schwannomas re-excited by uninjected bacteria.

[0114] To investigate whether intratumoral VNP20009 in schwannomas alone, or in combination with anti-PD-1 mAb, could generate a durable antitumor adaptive immune response, we used a re-compulsion model. 08031-9 mouse schwannomas were implanted into the left side of FVB / N mice, and as... Figure 5A schematic diagram shows the following groups: i) intratumoral injection of VNP20009, ii) intraperitoneal injection of anti-PD-1 mAb P, iii) intratumoral injection of VNP20009 and intraperitoneal injection of anti-PD-1 mAb, and iv) intratumoral injection of PBS. When the average tumor size reached 150 mm... 3 50 On day 8 post-implantation, VNP20009 was injected intratumorally (10 μL in 100 μL). 4 CFU). Anti-PD-1 mAb (250 μg / injection) was administered intraperitoneally on days 7, 10, 13, and 16 after tumor cell implantation. 51 Consistent with the results shown in Figure 3, all treatment regimens—VNP20009 / anti-PD-1 mAb, VNP20009, and anti-PD-1 mAb—inhibited tumor growth compared to PBS, and there was an additive effect when VNP20009 was combined with anti-PD-1 mAb. Figure 4B Twelve days after bacterial injection into the subcutaneous tumor (and 13 days after the first application of an immune checkpoint inhibitor), animals were re-stimulated by implanting 08031-9FC schwannomas into the contralateral sciatic nerve. The inventors chose the intra-sciatic location because it is in situ and because these allogeneic schwannomas develop more rapidly within the nerve than subcutaneously, thus facilitating observation of the effects. Intra-sciatic tumor growth was monitored by bioluminescence imaging, and tumor growth was inhibited in mice previously treated with VNP20009 or VNP20009 / anti-PD-1 mAb compared to the PBS control, with no difference between the two treatments. Figure 4D Compared to PBS, prior treatment with anti-PD-1 mAb alone did not alter tumor growth. Figure 4D It is noteworthy that the inhibitory effect of intratumoral VNP20009 is greater in re-excited tumors (). Figure 4D It appears to be more common in primary bacterial schwannomas than in those with primary bacterial injection. Figure 4B (It is larger in the middle.)

[0115] We again analyzed the T cell composition of injected tumors at the time of sacrifice and re-excited tumors by flow cytometry. In subcutaneous primary tumors, VNP20009 (8.47%), anti-PD-1 mAb (3.39%), and the combination of anti-PD-1 mAb and VNP20009 (9.39%) reduced the incidence of invasive CD4+ tumors compared to the PBS-injected control (0.99%). + The percentage of helper T cells increased; VNP20009 had a greater effect than anti-PD1-mAb, but compared with bacteria alone, there was no increase when checkpoint inhibitors were added to the bacteria. Figure 4CCompared with PBS injection (3.83%), VNP20009 (11.2%), anti-PD-1 mAb (6.69%), and the combination of VNP20009 and anti-PD-1 mAb (15.5%) also reduced CD8+ in subcutaneous tumors. + An increase in the percentage of cytotoxic T cells; in this case, the increase was greater in tumors treated with immune checkpoints than in tumors treated with injected bacteria, and the combination therapy resulted in a larger CD8 count compared to bacteria alone. + T cell induction ( Figure 4C In contrast, the proportion of CD25+ regulatory T cells in subcutaneous tumors was not altered by systemic anti-PD-1 mAb (11.2%) compared to the PBS control (12.2%), but was altered by intratumoral VNP20009 injection (9.52%) and the combination of bacterial injection and checkpoint inhibition (4.90%). Figure 4C Both inhibit it. The combined therapy has a greater inhibitory effect on tumor Tregs than bacterial therapy alone. Figure 4C ).

[0116] Quantitative analysis of T-lymphocyte populations in intraischial re-stimulation tumors revealed different therapeutic effects compared to primary subcutaneous tumors. Compared to PBS control animals (5.59%), CD4... + The percentage of helper T cells increased only in tumors of mice previously exposed to the combination of VNP20009 and anti-PD-1 mAb (13%); previous monotherapy with either VNP20009 (7.49%) or anti-PD-1 mAb (6.93%) had no effect. Figure 4E In these re-provoked tumors, the percentage of CD8+ cytotoxic T cells was increased in mice previously treated with VNP20009 (14.1%) or the VNP20009 / anti-PD-1 mAb combination (23.1%) compared to the PBS control (11.5%); previous checkpoint inhibition alone had no effect (anti-PD-1 mAb, 12.2%). Figure 4E Similarly, compared with PBS control mice (10.4%), previous VNP20009 (6.01%) or VNP20009 / anti-PD-1 mAb combination (3.82%) treatment reduced CD25 levels in re-exposed tumors. + The percentage of Treg decreased, but anti-PD-1 mAb did not reduce that percentage (8.16%). Figure 4E While previous immune checkpoint inhibition alone had no effect on the percentage of Tregs in re-excited schwannomas, the addition of anti-PD-1 mAb to VNP20009 enhanced the inhibitory effect of previous bacterial therapy alone. Figure 4E ).

[0117] Example 5. Intratumoral injection of Salmonella typhimurium inhibits angiogenesis in allogeneic rat schwannomas.

[0118] Salmonella typhimurium has been shown to be an important pro-angiogenic factor. 52 The expression of vascular endothelial growth factor (VEGF) is reduced, and it exhibits anti-angiogenic properties in preclinical cancer models. 24,53 Bevacizumab (an anti-angiogenic monoclonal antibody targeting VEGF-A) can control the growth of schwannomas in a subset of individuals with schwannomas. 54 Based on these observations, the inventors investigated the effect of intratumoral injection of attenuated Salmonella typhimurium on tumor angiogenesis.

[0119] Compared to a control group injected with PBS, inhibition of tumor angiogenesis was demonstrated in 08031-9 schwannomas of the ischial region of mice evaluated 2 weeks after intratumoral injection of attenuated Salmonella typhimurium. Tumor angiogenesis was assessed by direct observation and targeting the vascular endothelial marker CD31. + Immunohistochemistry to evaluate 55 ( Figure 5 Macroscopic evaluation of the tumors (N=6 mice / group) showed easily distinguishable differences between all schwannomas injected with PBS (bright red with prominent external blood vessels) and tumors injected with Salmonella typhimurium (pale in color with very little or no external angiogenesis). Figure 5 ). Compared with tumors injected with PBS (45.71±4.75; Figure 5 Compared to tumors injected with VNP20009 (N=3 / group), both tumors injected with ΔppGpp and tumors injected with ΔppGpp showed a reduction in CD31 counts. + Cells (7.71±1.89, P<0.001 and 8.41±1.68, P<0.001, respectively).

[0120] Example 6. Intratumoral injection of Salmonella typhimurium inhibits the growth of subcutaneous xenograft human NF1 tumors, sporadic human MPNST tumors, and human meningiomas.

[0121] We also evaluated the effects of intratumoral Salmonella typhimurium (VNP20009 and ΔppGpp) injection on the growth of a human NF-1 xenograft model in which immunocompromised nu / nu mice had human NF1-associated (S462TY) bacteria subcutaneously implanted on the left side. Figure 6A ) or sporadic (STS26, Figure 6B Malignant peripheral nerve sheath tumor cells (MPNST) 56 Furthermore, benign meningiomas (Ben-Men-1) were implanted in the nu / nu mice. Figure 6C) or malignant meningioma (CH-157MN, Figure 6D The efficacy of intratumoral Salmonella typhimurium was demonstrated in a xenograft model using subcutaneous implantation of a cell line. In the test model, mice were divided into three groups (n=5): VNP20009 or ΔppGpp (10 in 100 μl). 4 CFU was used as a control for PBS injection. Once the tumor mass was visible to the naked eye, intratumoral injection was performed, and tumor growth was monitored using calipers.

[0122] In an NF-1 xenograft model, the inventors' data showed that intratumoral injection of VNP20009 or ΔppGpp significantly inhibited tumor growth of NF-1-associated S462TY MPNST cells compared with intratumoral PBS injection (P<0.001). Figure 6A And it controlled the tumor growth of rapidly growing sporadic STS26T MPNST cells (P<0.001). Figure 6B At sacrifice (day 31 for S462TY and day 30 for STS26T), mice injected with VNP20009 and ΔppGpp showed approximately 7-fold and 4-fold reduction in tumor size, respectively, in the S462TY and STS26T models, compared to the PBS control.

[0123] Similarly, in a meningioma xenograft model, intratumoral injection of VNP20009 or ΔppGpp resulted in benign (Ben-Men-1) tumors being less likely to develop lesions compared to PBS controls. Figure 6C The tumor growth of the meningioma significantly regressed and malignancy was controlled (CH-157). Figure 6D Tumor growth of meningiomas. In the benign model, in mice injected with VNP20009 or ΔppGpp Salmonella typhimurium, 2 out of 5 mice showed complete regression on the day of sacrifice (day 38). On the day of sacrifice for malignant meningiomas, the tumor size in mice injected with VNP20009 and ΔppGpp was approximately 3 times smaller than that in the PBS control. The experiment was stopped on day 23 when tumors in the control group ulcerated / necrotized.

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[0204] Other implementation plans

[0205] It should be understood that although the invention has been described in detail therewith, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the appended claims.

Claims

1. Use of a composition comprising, optionally in combination with an immune checkpoint inhibitor and / or angiogenesis inhibitor, as an active ingredient, in the preparation of a medicament for treating a subject with a benign nervous system tumor by administering to the subject a therapeutically effective amount of the composition. The benign nervous system tumors mentioned above are neurofibromatosis 1 (NF1), neurofibromatosis 2 (NF2), or meningioma; The attenuated Salmonella is an attenuated strain of Salmonella Typhimurium, and the attenuated strain of Salmonella Typhimurium is a Salmonella Typhimurium serotype entero-Salmonella strain VNP20009 with modified lipid A (msbB–) and purine auxotrophic mutation (purI–).

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

3. The use according to claim 1, wherein the subject has a condition associated with an increased risk of benign nervous system tumors.

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

5. The use according to claim 1, wherein the composition does not contain Clostridium novie.

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

7. The use according to claim 1, wherein the checkpoint inhibitor is an inhibitor of PD-1 signaling or CTLA-4 signaling.

8. The use according to claim 7, wherein the inhibitor of PD-1 signaling is an antibody that binds to PD-1, CD40, PD-L1 or CTLA-4.

9. The use according to claim 1, wherein the angiogenesis inhibitor is an inhibitor of vascular endothelial growth factor VEGF or its receptor (VEGFR).

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

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