Development of an anti-tauopathy mucosal vaccine specifically targeting pathologic conformers.

Alzheimer's disease (AD) and related tauopathies are associated with pathological tau protein aggregation, which plays an important role in neurofibrillary degeneration and dementia. Targeted immunotherapy to eliminate pathological tau aggregates is known to improve cognitive deficits in AD animal models. The tau repeat domain (TauRD) plays a pivotal role in tau-microtubule interactions and is critically involved in the aggregation of hyperphosphorylated tau proteins. Because TauRD forms the structural core of tau aggregates, the development of immunotherapies that selectively target TauRD-induced pathological aggregates holds great promise for the modulation of tauopathies. In this study, we generated recombinant TauRD polypeptide that form neurofibrillary tangle-like structures and evaluated TauRD-specific immune responses following intranasal immunization in combination with the mucosal adjuvant FlaB. In BALB/C mice, repeated immunizations at one-week intervals induced robust TauRD-specific antibody responses in a TLR5-dependent manner. Notably, the resulting antiserum recognized only the aggregated form of TauRD, while ignoring monomeric TauRD. The antiserum effectively inhibited TauRD filament formation and promoted the phagocytic degradation of TauRD aggregate fragments by microglia. The antiserum also specifically recognized pathological tau conformers in the human AD brain. Based on these results, we engineered a built-in flagellin-adjuvanted TauRD (FlaB-TauRD) vaccine and tested its efficacy in a P301S transgenic mouse model. Mucosal immunization with FlaB-TauRD improved quality of life, as indicated by the amelioration of memory deficits, and alleviated tauopathy progression. Notably, the survival of the vaccinated mice was dramatically extended. In conclusion, we developed a mucosal vaccine that exclusively targets pathological tau conformers and prevents disease progression.

Engineered oncolytic bacteria HCS1 exerts high immune stimulation and safety profiles for cancer therapy.

Background and rationale: Attenuated Salmonella typhimurium VNP20009 has been used to treat tumor-bearing mice and entered phase I clinical trials. However, its mild anticancer effect in clinical trials may be related to insufficient bacterial colonization and notable adverse effects with increasing dosages. Guanosine 5'-diphosphate-3'-diphosphate (ppGpp) synthesis-deficient Salmonella is an attenuated strain with good biosafety and anticancer efficacy that has been widely investigated in various solid cancers in preclinical studies. Integration of the advantages of these two strains may provide a new solution for oncolytic bacterial therapy. Methods: We incorporated the features of ΔppGpp into VNP20009 and obtained the HCS1 strain by deleting relA and spoT, and then assessed its cytotoxicity in vitro and antitumor activities in vivo. Results: In vitro experiments revealed that the invasiveness and cytotoxicity of HCS1 to cancer cells were significantly lower than those of the VNP20009. Additionally, tumor-bearing mice showed robust cancer suppression when treated with different doses of HCS1 intravenously, and the survival time and cured mice were dramatically increased. Furthermore, HCS1 can increase the levels of pro-inflammatory cytokines in tumor tissues and relieve the immunosuppression in the tumor microenvironments. It can also recruit abundant immune cells into tumor tissues, thereby increasing immune activation responses. Conclusion: The newly engineered Salmonella HCS1 strain manifests high prospects for cancer therapeutics and is a promising option for future clinical cancer immunotherapy.

Synergistic cancer immunotherapy utilizing programmed Salmonella typhimurium secreting heterologous flagellin B conjugated to interleukin-15 proteins.

The use of appropriately designed immunotherapeutic bacteria is an appealing approach to tumor therapy because the bacteria specifically target tumor tissue and deliver therapeutic payloads. The present study describes the engineering of an attenuated strain of Salmonella typhimurium deficient in ppGpp biosynthesis (SAM) that could secrete Vibrio vulnificus flagellin B (FlaB) conjugated to human (hIL15/FlaB) and mouse (mIL15/FlaB) interleukin-15 proteins in the presence of L-arabinose (L-ara). These strains, named SAMphIF and SAMpmIF, respectively, secreted fusion proteins that retained bioactivity of both FlaB and IL15. SAMphIF and SAMpmIF inhibited the growth of MC38 and CT26 subcutaneous (sc) tumors in mice and increased mouse survival rate more efficiently than SAM expressing FlaB alone (SAMpFlaB) or IL15 alone (SAMpmIL15 and SAMphIL15), although SAMpmIF had slightly greater antitumor activity than SAMphIF. The mice treated with these bacteria showed enhanced macrophage phenotype shift, from M2-like to M1-like, as well as greater proliferation and activation of CD4+ T, CD8+ T, NK, and NKT cells in tumor tissues. After tumor eradication by these bacteria, ≥50% of the mice show no evidence of tumor recurrence upon rechallenge with the same tumor cells, indicating that they had acquired long-term immune memory. Treatment of mice of 4T1 and B16F10 highly malignant sc tumors with a combination of these bacteria and an immune checkpoint inhibitor, anti-PD-L1 antibody, significantly suppressed tumor metastasis and increased mouse survival rate. Taken together, these findings suggest that SAM secreting IL15/FlaB is a novel therapeutic candidate for bacterial-mediated cancer immunotherapy and that its antitumor activity is enhanced by combination with anti-PD-L1 antibody.

Exploring the potential of exosomes in diagnosis and drug delivery for pancreatic ductal adenocarcinoma.

Pancreatic cancer (PC) is a cancer of the digestive system, and pancreatic ductal adenocarcinoma (PDAC) accounts for approximately 90% of all PC cases. Exosomes derived from PDAC (PDAC-exosomes) promote PDAC development and metastasis. Exosomes are nanoscale vesicles secreted by most cells, which can carry biologically active molecules and mediate communication and cargo transportation among cells. Recent studies have focused on transforming exosomes into good drug delivery systems (DDSs) to improve the clinical treatment of PDAC. This review considers PDAC as the main research object to introduce the role of PDAC-exosomes in PDAC development and metastasis. This review focuses on the following two themes: (a) the great potential of PDAC-exosomes as new diagnostic markers for PDAC, and (b) the transformation of exosomes into potential DDSs.

Photodynamic therapy-improved oncolytic bacterial immunotherapy with FAP-encoding S. typhimurium.

Genetically engineered bacterial cancer therapy presents several advantages over conventional therapies. However, the anticancer effects of bacterium-based therapies remain insufficient, and serious side effects may be incurred with the increase in therapeutic dosages. Photodynamic therapy (PDT) suppresses tumor growth by producing reactive oxygen species (ROS) through specific laser-activated photosensitizers (PSs). Tumor-specific PS delivery and activatable ROS generation are two critical aspects for PDT advancement. Here, we propose PDT-enhanced oncolytic bacterial immunotherapy (OBI) by using genetically engineered avirulent Salmonella expressing a fluorogen-activating protein (FAP). Upon binding to a fluorogen, FAP could be activated and generate fluorescence and ROS. The instant activation of persistent fluorescence was detected in tumors through bacterium-based imaging. In a colon cancer model, PDT-OBI showed an enhanced tumor inhibition effect and prolonged animal survival. Mechanically, PDT generated ROS, resulting in the killing of cancer cells and over-accumulated bacteria. The pathogen-associated molecular patterns and damage-associated molecular patterns released from the destroyed bacteria and cancer cells recruited and activated immune cells (macrophages, neutrophils, and dendritic cells), which released additional proinflammatory cytokines (TNF-α and IL-1ß); reduced anti-inflammatory cytokines (IL-10); and further enhanced immune cell infiltration in a positive-feedback manner, thus reducing bacterium-induced side effects and improving anticancer activities. This synergistic therapy has promising potential for cancer immunotherapy.

Comparison of Anticancer Activities and Biosafety Between Salmonella enterica Serovar Typhimurium ΔppGpp and VNP20009 in a Murine Cancer Model.

Salmonella Typhimurium defective in guanosine 5'-diphosphate-3'-diphosphate (ppGpp) synthesis (ΔppGpp) is an attenuated strain with good biosafety and excellent anticancer efficacy. It has been widely applied in preclinical studies of anticancer therapy for various types of solid cancer. VNP20009 is another genetically modified auxotrophic strain with 108-kb deletion, purI- , msbB- , and many single nucleotide polymorphisms (SNPs); it has shown promising therapeutic efficacy in various preclinical tumor models and entered phase I clinical trials. Here, the invasion activities and virulence of ΔppGpp were obviously lower than those of the VNP20009 strain when tested with cancer cells in vitro. In addition, the MC38 tumor-bearing mice showed comparable cancer suppression when treated with ΔppGpp or VNP20009 intravenously. However, the ΔppGpp-treated mice showed 16.7% of complete cancer eradication and prolonged survival in mice, whereas VNP20009 showed higher toxicity to animals, even with equal tumor size individually. Moreover, we found decreased levels of inflammatory cytokines in circulation but strengthened immune boost in tumor microenvironments of ΔppGpp-treated mice. Therefore, the engineered ΔppGpp has high potential for cancer therapeutics, and it is a promising option for future clinical cancer therapy.

Visualized Cancer Immunotherapy with Engineered Salmonella typhimurium.

Genetically engineered Salmonella typhimurium can specifically colonize tumor tissues and drastically inhibit tumor growth. Vibrio vulnificus flagellin B (FlaB), a natural ligand of Toll-like receptor 5 (TLR5) that can activate robust host immune system, is an excellent adjuvant for cancer immunotherapy with high binding affinity to TLR5. Here, we constructed attenuated S. typhimurium that expresses flagellin B (FlaB) with a controlled expression system to enhance targeted cancer immunotherapy with increased good safety profiles. Visualized therapy can also be achieved with bioluminescence imaging by introducing the lux operon into the attenuated Salmonella.

Targeting of pancreatic cancer cells and stromal cells using engineered oncolytic Salmonella typhimurium.

Pancreatic cancer is resistant to conventional therapeutic interventions, mainly due to abundant cancer stromal cells and poor immune cell infiltration. Here, we used a targeted cancer therapy approach based on attenuated Salmonella typhimurium engineered to express cytolysin A (ClyA) to target cancer stromal cells and cancer cells and treat pancreatic cancer in mice. Nude mice bearing subcutaneous or orthotopic human pancreatic cancers were treated with engineered S. typhimurium expressing ClyA. The tumor microenvironment was monitored to analyze stromal cell numbers, stromal cell marker expression, and immune cell infiltration. The attenuated bacteria accumulated and proliferated specifically in tumor tissues after intravenous injection. The bacteria secreted ClyA into the tumor microenvironment. A single dose of ClyA-expressing Salmonella markedly inhibited growth of pancreatic cancer both in subcutaneous xenograft- and orthotopic tumor-bearing nude mice. Histological analysis revealed a marked decrease in expression of stromal cell markers and increased immune cell (neutrophils and macrophages) infiltration into tumors after colonization by ClyA-expressing bacteria. ClyA-expressing S. typhimurium destroyed cancer stromal cells and cancer cells in mouse models of human pancreatic cancer. This approach provides a novel strategy for combining anticancer and anti-stromal therapy to treat pancreatic cancer.

Genetically engineered oncolytic bacteria as drug delivery systems for targeted cancer theranostics.

Drug delivery systems based on genetically engineered oncolytic bacteria have properties that cannot be achieved by traditional therapeutic interventions. Thus, they have attracted considerable attention in cancer therapies. Attenuated bacteria can specifically target and actively penetrate tumor tissues and play an important role in cancer suppression as the "factories" of diverse anticancer drugs. Over the past decades, several bacterial strains including Salmonella and Clostridium have been shown to effectively retard tumor growth and metastasis, and thus improve survival in preclinical models or clinical cases. In this review, we summarize the unique properties of oncolytic bacteria and their anticancer mechanisms and highlight the particular advantages compared with traditional strategies. With the current research progress, we demonstrate the potential value of oncolytic bacteria-based drug delivery systems for clinical applications. In addition, we discuss novel strategies of cancer therapies integrating oncolytic bacteria, which will provide hope to further improve and standardize the current regimens in the near future.

RBM38 in cancer: role and mechanism.

Cancer is the second leading cause of death globally. Abnormity in gene expression regulation characterizes the trajectory of tumor development and progression. RNA-binding proteins (RBPs) are widely dysregulated, and thus implicated, in numerous human cancers. RBPs mainly regulate gene expression post-transcriptionally, but emerging studies suggest that many RBPs can impact transcription by acting on chromatin as transcription factors (TFs) or cofactors. Here, we review the evidence that RBM38, an intensively studied RBP, frequently plays a tumor-suppressive role in multiple human cancer types. Genetic studies in mice deficient in RBM38 on different p53 status also establish RBM38 as a tumor suppressor (TS). By uncovering a spectrum of transcripts bound by RBM38, we discuss the diversity in its mechanisms of action in distinct biological contexts. Examination of the genomic features and expression pattern of RBM38 in human tissues reveals that it is generally lost but rarely mutated, in cancers. By assessing future trends in the study of RBM38 in cancer, we signify the possibility of targeting RBM38 and its related pathways as therapeutic strategies against cancer.

In Vivo Bioluminescence Imaging for Targeting Acute Hypoxic/Ischemic Small Intestine with Engineered Salmonella typhimurium.

This study aimed at investigating the feasibility of bioluminescence imaging (BLI) with engineered Salmonella typhimurium (ΔppGpp S. typhimurium) for visualizing acute hypoxic/ischemic bowels. At the start of 12- or 24-h reperfusion, ΔppGpp S. typhimurium was injected into the lateral tail veins of rats in which three segments of the small intestine were respectively subjected to 2, 3, and 4 h of ischemia. BLI and magnetic resonance imaging were performed at each reperfusion time point. Bioluminescence was exclusively detected in the hypoxic/ischemic segment of the intestine, showing the ability of ΔppGpp S. typhimurium to specifically target and proliferate in a hypoxic/ischemic area. Serial monitoring of these rat models revealed a progressive increase in bacterial bioluminescence in the ischemic intestines in conjunction with viable bacterial counts. The viable bacterial counts were positively correlated with lactate dehydrogenase levels after 24 h of reperfusion following 3 or 4 h of ischemia as well as interleukin-6 levels after 24 h of reperfusion following 4 h of ischemia. Our findings demonstrated that BLI was able to detect the acute hypoxic/ischemic bowel via monitoring of the distribution, internalization, and activity of administered ΔppGpp S. typhimurium. These findings may be useful for the early diagnosis of ischemic bowel disease.

Targeted cancer immunotherapy with genetically engineered oncolytic Salmonella typhimurium.

Conventional chemotherapies have some limitations, including the lack of selectivity, high toxicity to normal tissues, multidrug resistance, and tumor relapse. Recently, great progress was made in immunotherapies for anticancer research, with bacteria-mediated cancer therapy one of the most promising approaches among them. Attenuated Salmonella have very specific targeting to various solid cancers, making them ideal vectors for the delivery and expression of immunostimulators. They have native bacterial immunogenicity and induce strong anticancer immunity in vivo. In this review, the recent advances in Salmonella-mediated cancer immunotherapies and the related mechanisms of Salmonella-based cancer therapies are summarized.

A Fusion Protein of Derp2 Allergen and Flagellin Suppresses Experimental Allergic Asthma.

PURPOSE: The house dust mite (HDM) is one of the most important sources of indoor allergens and a significant cause of allergic rhinitis and allergic asthma. Our previous studies demonstrated that Vibrio vulnificus flagellin B (FlaB) plus allergen as a co-treatment mixture improved lung function and inhibited eosinophilic airway inflammation through the Toll-like receptor 5 signaling pathway in an ovalbumin (OVA)- or HDM-induced mouse asthma model. In the present study, we fused the major mite allergen Derp2 to FlaB and compared the therapeutic effects of the Derp2-FlaB fusion protein with those of a mixture of Derp2 and FlaB in a Derp2-induced mouse asthma model. METHODS: BALB/c mice sensitized with Derp2 + HDM were treated with Derp2, a Derp2 plus FlaB (Derp2 + FlaB) mixture, or the Derp2-FlaB fusion protein 3 times at 1-week intervals. Seven days after the final treatment, the mice were challenged intranasally with Derp2, and airway responses and Derp2-specific immune responses were evaluated. RESULTS: The Derp2-FlaB fusion protein was significantly more efficacious in reducing airway hyperresponsiveness, lung eosinophil infiltration, and Derp2-specific IgE than the Derp2 + FlaB mixture. CONCLUSIONS: The Derp2-FlaB fusion protein showed a strong anti-asthma immunomodulatory capacity, leading to the prevention of airway inflammatory responses in a murine disease model through the inhibition of Th2 responses. These findings suggest that the Derp2-FlaB fusion protein would be a promising vaccine candidate for HDM-mediated allergic asthma therapy.

Genetically-engineered Salmonella typhimurium expressing TIMP-2 as a therapeutic intervention in an orthotopic glioma mouse model.

Glioma is one of the most devastating and refractory cancers. The main factors underlying therapeutic failure include extremely invasive characteristics and lack of effective methods for drug delivery. Attenuated Salmonella strains presented a high concentration of tumor targets in various types of cancer models, suggesting a role as potential vectors for drug delivery. In this study, we genetically engineered an attenuated strain of Salmonella as an anti-invasive vector for the targeted delivery and expression of tissue inhibitor of metalloproteinases 2 (TIMP-2) in an orthotopic nude mouse model of glioma. The bioluminescence signals related to tumor size significantly declined in the TIMP-2-expressing Salmonella (SLpTIMP-2)-treated group compared with the control group. Compared with the control group with a survival rate of an average of 33 days, the SLpTIMP-2 group showed an extended survival rate by nearly 60% and lasted an average period of 53 days with TIMP-2 induction. These results indicated the promising therapeutic potential of S. typhimurium for targeted delivery and secretion of TIMP-2 in glioma.

A High-Affinity Repebody for Molecular Imaging of EGFR-Expressing Malignant Tumors.

The accurate detection of disease-related biomarkers is crucial for the early diagnosis and management of disease in personalized medicine. Here, we present a molecular imaging of human epidermal growth factor receptor (EGFR)-expressing malignant tumors using an EGFR-specific repebody composed of leucine-rich repeat (LRR) modules. The repebody was labeled with either a fluorescent dye or radioisotope, and used for imaging of EGFR-expressing malignant tumors using an optical method and positron emission tomography. Our approach enabled visualization of the status of EGFR expression, allowing quantitative evaluation in whole tumors, which correlated well with the EGFR expression levels in mouse or patients-derived colon cancers. The present approach can be effectively used for the accurate detection of EGFR-expressing cancers, assisting in the development of a tool for detecting other disease biomarkers.

Two-step enhanced cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous flagellin.

We report a method of cancer immunotherapy using an attenuated Salmonella typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues. Engineered FlaB-secreting bacteria effectively suppressed tumor growth and metastasis in mouse models and prolonged survival. By using Toll-like receptor 5 (TLR5)-negative colon cancer cell lines, we provided evidence that the FlaB-mediated tumor suppression upon bacterial colonization is associated with TLR5-mediated host reactions in the tumor microenvironment. These therapeutic effects were completely abrogated in TLR4 and MyD88 knockout mice, and partly in TLR5 knockout mice, indicating that TLR4 signaling is a requisite for tumor suppression mediated by FlaB-secreting bacteria, whereas TLR5 signaling augmented tumor-suppressive host reactions. Tumor microenvironment colonization by engineered Salmonella appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonella resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Together, these findings provide evidence that nonvirulent tumor-targeting bacteria releasing multiple TLR ligands can be used as cancer immunotherapeutics.

Targeted Cancer Therapy Using Engineered Salmonella typhimurium.

Obligate or facultative anaerobic bacteria such as Bifidobacterium, Clostridium, Salmonella, or Escherichia coli specifically colonize and proliferate inside tumor tissues and inhibit tumor growth. Among them, attenuated Salmonella typhimurium (S. typhimurium) has been widely studied in animal cancer models and Phase I clinical trials in human patients. S. typhimurium genes are easily manipulated; thus diverse attenuated strains of S. typhimurium have been designed and engineered as tumor-targeting therapeutics or drug delivery vehicles that show both an excellent safety profile and therapeutic efficacy in mouse models. An attenuated strain of S. typhimurium, VNP20009, successfully targeted human metastatic melanoma and squamous cell carcinoma in Phase I clinical trials; however, the efficacy requires further refinement. Along with the characteristics of self-targeting, proliferation, and deep tissue penetration, the ease of genetic manipulation allows for the production of more attenuated strains with greater safety profiles and vector systems that deliver designable cargo molecules for cancer diagnosis and/or therapy. Here, we discuss recent progress in the field of Salmonellae-mediated cancer therapy.

RGD Peptide Cell-Surface Display Enhances the Targeting and Therapeutic Efficacy of Attenuated Salmonella-mediated Cancer Therapy.

Bacteria-based anticancer therapies aim to overcome the limitations of current cancer therapy by actively targeting and efficiently removing cancer. To achieve this goal, new approaches that target and maintain bacterial drugs at sufficient concentrations during the therapeutic window are essential. Here, we examined the tumor tropism of attenuated Salmonella typhimurium displaying the RGD peptide sequence (ACDCRGDCFCG) on the external loop of outer membrane protein A (OmpA). RGD-displaying Salmonella strongly bound to cancer cells overexpressing αvß3, but weakly bound to αvß3-negative cancer cells, suggesting the feasibility of displaying a preferential homing peptide on the bacterial surface. In vivo studies revealed that RGD-displaying Salmonellae showed strong targeting efficiency, resulting in the regression in αvß3-overexpressing cancer xenografts, and prolonged survival of mouse models of human breast cancer (MDA-MB-231) and human melanoma (MDA-MB-435). Thus, surface engineering of Salmonellae to display RGD peptides increases both their targeting efficiency and therapeutic effect.

Salmonella typhimurium Suppresses Tumor Growth via the Pro-Inflammatory Cytokine Interleukin-1ß.

Although strains of attenuated Salmonella typhimurium and wild-type Escherichia coli show similar tumor-targeting capacities, only S. typhimurium significantly suppresses tumor growth in mice. The aim of the present study was to examine bacteria-mediated immune responses by conducting comparative analyses of the cytokine profiles and immune cell populations within tumor tissues colonized by E. coli or attenuated Salmonellae. CT26 tumor-bearing mice were treated with two different bacterial strains: S. typhimurium defective in ppGpp synthesis (ΔppGpp Salmonellae) or wild-type E. coli MG1655. Cytokine profiles and immune cell populations in tumor tissue colonized by these two bacterial strains were examined at two time points based on the pattern of tumor growth after ΔppGpp Salmonellae treatment: 1) when tumor growth was suppressed ('suppression stage') and 2) when they began to re-grow ('re-growing stage'). The levels of IL-1ß and TNF-α were markedly increased in tumors colonized by ΔppGpp Salmonellae. This increase was associated with tumor regression; the levels of both IL-1ß and TNF-α returned to normal level when the tumors started to re-grow. To identify the immune cells primarily responsible for Salmonellae-mediated tumor suppression, we examined the major cell types that produce IL-1ß and TNF-α. We found that macrophages and dendritic cells were the main producers of TNF-α and IL-1ß. Inhibiting IL-1ß production in Salmonellae-treated mice restored tumor growth, whereas tumor growth was suppressed for longer by local administration of recombinant IL-1ß or TNF-α in conjunction with Salmonella therapy. These findings suggested that IL-1ß and TNF-α play important roles in Salmonella-mediated cancer therapy. A better understanding of host immune responses in Salmonella therapy may increase the success of a given drug, particularly when various strategies are combined with bacteriotherapy.