Jin Hai Zheng

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

Engineered Salmonella secreting heterologous bacterial flagellin suppress tumor growth by activating intratumoral macrophages. Two bacteria can be better than one In some cases, injecting tumors with specific bacteria can help eradicate the tumors by stimulating inflammation and triggering an antitumor immune response. A classic example of this is injection of bladder cancer with bacillus Calmette-Guérin, but more recent approaches have used bacteria such as Clostridium and Salmonella species. Building on the idea of antitumor bacterial therapy, Zheng et al. engineered a weakened strain of Salmonella typhimurium to produce the flagellin B protein from another bacterium, Vibrio vulnificus. The engineered bacteria induced an effective antitumor immune response, successfully treating tumors in several different mouse models with no evidence of toxicity. 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.

Engineering of Bacteria for the Visualization of Targeted Delivery of a Cytolytic Anticancer Agent

A number of recent reports have demonstrated that attenuated Salmonella typhimurium are capable of targeting both primary and metastatic tumors. The use of bacteria as a vehicle for the delivery of anticancer drugs requires a mechanism that precisely regulates and visualizes gene expression to ensure the appropriate timing and location of drug production. To integrate these functions into bacteria, we used a repressor-regulated tetracycline efflux system, in which the expression of a therapeutic gene and an imaging reporter gene were controlled by divergent promoters (tetAP and tetRP) in response to extracellular tetracycline. Attenuated S. typhimurium was transformed with the expression plasmids encoding cytolysin A, a therapeutic gene, and renilla luciferase variant 8, an imaging reporter gene, and administered intravenously to tumor-bearing mice. The engineered Salmonella successfully localized to tumor tissue and gene expression was dependent on the concentration of inducer, indicating the feasibility of peripheral control of bacterial gene expression. The bioluminescence signal permitted the localization of gene expression from the bacteria. The engineered bacteria significantly suppressed both primary and metastatic tumors and prolonged survival in mice. Therefore, engineered bacteria that carry a therapeutic and an imaging reporter gene for targeted anticancer therapy can be designed as a theranostic agent.

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 αvβ3, but weakly bound to αvβ3-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 αvβ3-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.

Comparison of 18F-Labeled Fluoroalkylphosphonium Cations with 13N-NH3 for PET Myocardial Perfusion Imaging

Despite substantial advances in the diagnosis of cardiovascular disease, there is a need for 18F-labeled myocardial perfusion agents for the diagnosis of ischemic heart disease because current PET tracers for myocardial perfusion imaging have a short half-life that limits their widespread clinical use in PET. Thus, 18F-labeled fluoroalkylphosphonium derivatives (18F-FATPs), including (5-18F-fluoropentyl)triphenylphosphonium cation (18F-FPTP), (6-18F-fluorohexyl)triphenylphosphonium cation (18F-FHTP), and (2-(2-18F-fluoroethoxy)ethyl)triphenylphosphonium cation (18F-FETP), were synthesized. The myocardial extraction and image quality of the 18F-FATPs were compared with those of 13N-NH3 in rat models. Methods: The first-pass extraction fraction (EF) values of the 18F-FATPs (18F-FPTP, 18F-FHTP, 18F-FETP) and 13N-NH3 were measured in isolated rat hearts perfused with the Langendorff method (flow velocities, 0.5, 4.0, 8.0, and 16.0 mL/min). Normal and myocardial infarction rats were imaged with small-animal PET after intravenous injection of 37 MBq of 18F-FATPs and 13N-NH3. To determine pharmacokinetics, a region of interest was drawn around the heart, and time–activity curves of the 18F-FATPs and 13N-NH3 were generated to obtain the counts per pixel per second. Defect size was analyzed on the basis of polar map images of 18F-FATPs and 13N-NH3. Results: The EF values of 18F-FATPs and 13N-NH3 were comparable at low flow velocity (0.5 mL/min), whereas at higher flows EF values of 18F-FATPs were significantly higher than those of 13N-NH3 (4.0, 8.0, and 16.0 mL/min, P < 0.05). Myocardium-to-liver ratios of 18F-FPTP, 18F-FHTP, 18F-FETP, and 13N-NH3 were 2.10 ± 0.30, 4.36 ± 0.20, 3.88 ± 1.03, and 0.70 ± 0.09, respectively, 10 min after injection, whereas myocardium-to-lung ratios were 5.00 ± 0.25, 4.33 ± 0.20, 7.98 ± 1.23, and 2.26 ± 0.14, respectively. Although 18F-FATPs and 13N-NH3 sharply delineated myocardial perfusion defects, defect size on the 13N-NH3 images was significantly smaller than on the 18F-FATP images soon after tracer injection (0–10 min, P = 0.027). Conclusion: 18F-FATPs exhibit higher EF values and more rapid clearance from the liver and lung than 13N-NH3 in normal rats, which led to excellent image quality in a rat model of coronary occlusion. Therefore, 18F-FATPs are promising new PET radiopharmaceuticals for myocardial perfusion imaging.

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.

Rhodobacter sphaeroides, a novel tumor-targeting bacteria that emits natural near-infrared fluorescence

Several optical imaging techniques have been used to monitor bacterial tropisms for cancer. Most such techniques require genetic engineering of the bacteria to express optical reporter genes. This study investigated a novel tumor‐targeting strain of bacteria, Rhodobacter sphaeroides 2.4.1 (R. sphaeroides), which naturally emits near‐infrared fluorescence, thereby facilitating the visualization of bacterial tropisms for cancer. To determine the penetration depth of bacterial fluorescence, various numbers of cells (from 108 to 1010 CFU) of R. sphaeroides and two types of Escherichia coli, which stably express green fluorescent protein (GFP) or red fluorescent protein (RFP), were injected s.c. or i.m. into mice. Bacterial tropism for cancer was determined after i.v. injection of R. sphaeroides (108 CFU) into mice implanted s.c. with eight types of tumors. The intensity of the fluorescence signal in deep tissue (muscle) from R. sphaeroides was much stronger than from E. coli‐expressing GFP or RFP. The near‐infrared fluorescence signal from R. sphaeroides was visualized clearly in all types of human or murine tumors via accumulation of bacteria. Analyses of C‐reactive protein and procalcitonin concentrations and body weights indicated that i.v. injection of R. sphaeroides does not induce serious systemic immune reactions. This study suggests that R. sphaeroides could be used as a tumor‐targeting microorganism for the selective delivery of drugs to tumor tissues without eliciting a systemic immune reaction and for visualizing tumors.

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.

Abstract 1610: Targeted cancer immunotherapy with engineeredSalmonella typhimuriumsecreting heterologous bacterial flagellin

Salmonella typhimurium is a facultative anaerobic bacteria and can selectively grow in tumors following systemic administration. Different strategies have been used to deliver payloads to tumor tissues using engineered Salmonellae. We report a novel method of cancer immunotherapy using an attenuated S. typhimurium strain engineered to secrete Vibrio vulnificus flagellin B (FlaB) in tumor tissues.Previously, we developed an attenuated strain of S. typhimurium, which is defective in ppGpp synthesis (ΔppGpp S. typhimurium), and manifested significantly increased LD50 by 100,000 to 1,000,000-fold. To generate an inducible vector system for bacterial expression of the therapeutic gene, we cloned the flaB gene into the pBAD plasmid vector in which the pelB leader sequence was cloned upstream of flaB to guide extracellular secretion; gene expression from the plasmid (pFlaB) was induced only in the presence of L-arabinose. To evaluate the antitumor activity of engineered S. typhimurium, C57BL/6 mice transplanted with MC38 tumors were injected intravenously (i.v.) with PBS, ΔppGpp S. typhimurium carrying an empty vector, or ΔppGpp S. typhimurium carrying pFlaB (+/− L-arabinose induction). To test whether the FlaB-secreting ΔppGpp S. typhimurium has inhibitory effects on human metastatic cancer, we next implanted HCT116-luc2 tumors into the intestinal wall of BALB/c athymic nu-/nu- mice using a surgical orthotopic implantation (SOI) procedure. Engineered FlaB-secreting Salmonellae significantly suppressed tumor growth and metastasis in mouse models and prolonged survival. By using TLR5-negative colon cancer cell lines (MC38 and HCT116), we have proved that the FlaB-mediated tumor suppression upon bacterial colonization should be 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, strongly suggesting that TLR4 signaling is a requisite for the FlaB-secreting bacteria-mediated tumor suppression where TLR5 signaling augmented tumor suppressive host reactions. Tumor colonization by engineered Salmonellae appeared to induce the infiltration of abundant immune cells such as monocytes/macrophages and neutrophils via the TLR4 signaling. Subsequent secretion of FlaB from colonizing Salmonellae resulted in phenotypic and functional activation of intratumoral macrophages with M1 phenotypes and a reciprocal reduction in M2-like suppressive activities. Taken together, these findings provide evidence that non-virulent tumor targeting bacteria liberating multiple TLR ligands can be used as novel cancer immunotherapeutics. Citation Format: Jung-Joon Min, Jin Hai Zheng, Yeongjin Hong, Hyon E. Choy, Joon Haeng Rhee. Targeted cancer immunotherapy with engineered Salmonella typhimurium secreting heterologous bacterial flagellin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1610. doi:10.1158/1538-7445.AM2017-1610

Abstract 4212: Molecular imaging of EGFR-expressing tumors with novel targeted protein scaffold, anti-EGFR repebody

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Repebody is a binding scaffold based on variable lymphocyte receptors which are nonimmunoglobulin antibodies composed of leucine-rich repeat modules in jawless vertebrates. Repebody can be developed against variety of epitopes by module engineering. In this study, EGFR-specific repebody (RBEGFR) was developed to visualize the status of receptor expression and to prevent ligand binding that may inhibit autophosphorylation and downstream intracellular signaling. We developed RBEGFR by phage display. H1650, HCC827, A549 (human non-small cell lung cancer) and HT29 (human colon cancer) were selected as EGFR expressing cell lines. MDA-MB-435 (human melanoma) and SW620 (human colon cancer) was selected as a negative control. Specific binding of RBEGFR to cells and cancer tissue was determined by immunofluorescence (IF) staining and/or FACS analysis. In vivo imaging was done by i.v. injection of FNR-675 labeled RBEGFR (30 μg/mouse) or 64Cu-NOTA- RBEGFR (7.4 MBq/mouse) in H1650, HCC827 and HT29-bearing mouse models using cooled CCD camera or microPET, respectively. Orthotopic colon cancer mice were generated by i.p. injection of Azoxymethane (AOM; 10 mg/kg) and oral administration of 2% dextran sulfate sodium (DSS). In vivo imaging was done by i.v. injection FNR-675 labeled RBEGFR (30 μg/mouse) in AOM/DSS mouse models using cooled CCD camera or fluorescence microendoscopy. In vitro and in vivo IF staining demonstrated that strong binding of RBEGFR to H1650, HCC827 and HT29, but not to MDA-MB-435 and SW620. In vivo near infrared (NIR) imaging demonstrated specific targeting of FNR-675-RBEGFR to grafted H1650, HCC827 and HT29 tumor in mice. The 64Cu-NOTA- RBEGFR was detected at the implanted tumor from 1 h (SUVmax: 1.34±0.12) after the injection, peaked at 6 h (1.75±0.18), maintained to 24 h (1.33±0.17). The radioactivity significantly decreased by blocking with cold form of 50 μM naive RBEGFR 1 day before injection of 64Cu-NOTA- RBEGFR, indicating specific binding of RBEGFR to EGFR in vivo. Optical NIR imaging after i.v. injection of FNR-675-RBEGFR showed specific signal in the abdomen of AOM/DSS mice, but not in control mice. Correlation with surgical/necropsy imaging, fluorescence endoscopy and pathology revealed strong accumulation of FNR-675-RBEGFR in malignant dysplasia, but weak or no accumulation in low grade tumor or benign lesion. In conclusion, the RBEGFR could be developed for specific targeting of cancer overexpressing EGFR. The fluorescence-labeled RBEGFR could be developed for imaging agent for detecting colonic dysplasia and assessing EGFR status. In particular, this agent may have a potential as an imaging companion diagnostics to predict therapeutic outcome of targeted therapy with monoclonal antibody for EGFR through pre-therapeutic visualization of EGFR status. Our work provides a basis to develop potential strategy of targeted immune-detection of cancers which may replace monoclonal antibodies. Citation Format: Misun Yun, Dong-Yeon Kim, Hyeon-Sik Kim, Jin Hai Zheng, AYoung Pyo, Jung-Joon Min. Molecular imaging of EGFR-expressing tumors with novel targeted protein scaffold, anti-EGFR repebody. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4212.

Abstract 4219: Surface-displayed RGD enhanced the targeting and therapeutic efficacy of attenuated Salmonella typhimurium

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Anticancer therapy using engineered bacteria aims to overcome limitations of current cancer therapy by actively targeting and efficiently removing cancer. In order to achieve this goal, new approaches are essential to target therapeutically resistant regions of tumors, to maintain enough number of bacteria in tumor and to deliver drugs at sufficient concentrations during the period of therapeutic process. Therefore, it is necessary to enhance the bacterial targeting efficiency through bacterial surface engineering. Here, we demonstrate that Salmonella tumor tropism can be strengthened significantly via surface displaying of RGD peptide sequence (ACDCRGDCFCG; RGD sequence) in the external loop of outer membrane protein A (OmpA) of attenuated Salmonella typhimurium. RGD-displaying Salmonella strongly bound to αvβ3 over-expressing cancer cells while showed weak binding to non-expressing cancer cells, indicating the feasibility of surface display with preferential homing peptide. In vivo bioluminescence imaging showed strong targeting efficiency of RGD-displayed Salmonella in αvβ3 over-expressing cancer xenografts (MDA-MB-231, MDA-MB-435, M21, and U87MG). The surface engineered bacteria significantly suppressed both human breast tumor (MDA-MB-231) and human melanoma (MDA-MB-435), and prolonged survival in mice. In conclusion, engineered bacteria displaying RGD peptides on the surface could advance both targeting efficiency and therapeutic effects. Citation Format: Seung-Hwan Park, Jin Hai Zheng, Hee-Seung Yun, In-Kyu Park, Yeongjin Hong, Hyun E. Choy, Jung-Joon Min. Surface-displayed RGD enhanced the targeting and therapeutic efficacy of attenuated Salmonella typhimurium. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4219.