MEndoB, a chimeric lysin featuring a novel domain architecture and superior activity for the treatment of staphylococcal infections.

Bacterial infections are a growing global healthcare concern, as an estimated annual 4.95 million deaths are associated with antimicrobial resistance (AMR). Methicillin-resistant Staphylococcus aureus is one of the deadliest pathogens and a high-priority pathogen according to the World Health Organization. Peptidoglycan hydrolases (PGHs) of phage origin have been postulated as a new class of antimicrobials for the treatment of bacterial infections, with a novel mechanism of action and no known resistances. The modular architecture of PGHs permits the creation of chimeric PGH libraries. In this study, the chimeric enzyme MEndoB was selected from a library of staphylococcal PGHs based on its rapid and sustained activity against staphylococci in human serum. The benefit of the presented screening approach was illustrated by the superiority of MEndoB in a head-to-head comparison with other PGHs intended for use against staphylococcal bacteremia. MEndoB displayed synergy with antibiotics and rapid killing in human whole blood with complete inhibition of re-growth over 24 h at low doses. Successful treatment of S. aureus-infected zebrafish larvae with MEndoB provided evidence for its in vivo effectiveness. This was further confirmed in a lethal systemic mouse infection model in which MEndoB significantly reduced S. aureus loads and tumor necrosis factor alpha levels in blood in a dose-dependent manner, which led to increased survival of the animals. Thus, the thorough lead candidate selection of MEndoB resulted in an outstanding second-generation PGH with in vitro, ex vivo, and in vivo results supporting further development.IMPORTANCEOne of the most pressing challenges of our era is the rising occurrence of bacteria that are resistant to antibiotics. Staphylococci are prominent pathogens in humans, which have developed multiple strategies to evade the effects of antibiotics. Infections caused by these bacteria have resulted in a high burden on the health care system and a significant loss of lives. In this study, we have successfully engineered lytic enzymes that exhibit an extraordinary ability to eradicate staphylococci. Our findings substantiate the importance of meticulous lead candidate selection to identify therapeutically promising peptidoglycan hydrolases with unprecedented activity. Hence, they offer a promising new avenue for treating staphylococcal infections.

A novel surface functionalization platform to prime extracellular vesicles for targeted therapy and diagnostic imaging.

Extracellular vesicles (EVs), nanovesicles released by cells to effectively exchange biological information, are gaining interest as drug delivery system. Yet, analogously to liposomes, they show short blood circulation times and accumulation in the liver and the spleen. For tissue specific delivery, EV surfaces will thus have to be functionalized. We present a novel platform for flexible modification of EVs with target-specific ligands based on the avidin-biotin system. Genetic engineering of donor cells with a glycosylphosphatidylinositol-anchored avidin (GPI-Av) construct allows the isolation of EVs displaying avidin on their surface, functionalized with any biotinylated ligand. For proof of concept, GPI-Av EVs were modified with i) a biotinylated antibody or ii) de novo designed and synthesized biotinylated ligands binding carbonic anhydrase IX (CAIX), a membrane associated enzyme overexpressed in cancer. Functionalized EVs showed specific binding and uptake by CAIX-expressing cells, demonstrating the power of the system to prepare EVs for cell-specific drug delivery.

Hypoxia Induces a HIF-1-Dependent Transition from Collective-to-Amoeboid Dissemination in Epithelial Cancer Cells.

Cancer metastases arise from a multi-step process that requires metastasizing tumor cells to adapt to signaling input from varying tissue environments [1]. As an early metastatic event, cancer cell dissemination occurs through different migration programs, including multicellular, collective, and single-cell mesenchymal or amoeboid migration [2-4]. Migration modes can interconvert based on changes in cell adhesion, cytoskeletal mechanotransduction [5], and/or proteolysis [6], most likely under the control of transcriptional programs such as the epithelial-to-mesenchymal transition (EMT) [7, 8]. However, how plasticity of tumor cell migration and EMT is spatiotemporally controlled and connected upon challenge by the tumor microenvironment remains unclear. Using 3D cultures of collectively invading breast and head and neck cancer spheroids, here we identify hypoxia, a hallmark of solid tumors [9], as an inducer of the collective-to-amoeboid transition (CAT), promoting the dissemination of amoeboid-moving single cells from collective invasion strands. Hypoxia-induced amoeboid detachment was driven by hypoxia-inducible factor 1 (HIF-1), followed the downregulation of E-cadherin, and produced heterogeneous cell subsets whose phenotype and migration were dependent (∼30%) or independent (∼70%) of Twist-mediated EMT. EMT-like and EMT-independent amoeboid cell subsets showed stable amoeboid movement over hours as well as leukocyte-like traits, including rounded morphology, matrix metalloproteinase (MMP)-independent migration, and nuclear deformation. Cancer cells undergoing pharmacological stabilization of HIFs retained their constitutive ability for early metastatic seeding in an experimental model of lung metastasis, indicating that hypoxia-induced CAT enhances cell release rather than early organ colonization. Induced by metabolic challenge, amoeboid movement may thus constitute a common endpoint of both EMT-dependent and EMT-independent cancer dissemination programs.

A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.

The complexity of the tumor microenvironment is difficult to mimic in vitro, particularly regarding tumor-host interactions. To enable better assessment of cancer immunotherapy agents in vitro, we developed a three-dimensional (3D) heterotypic spheroid model composed of tumor cells, fibroblasts, and immune cells. Drug targeting, efficient stimulation of immune cell infiltration, and specific elimination of tumor or fibroblast spheroid areas were demonstrated following treatment with a novel immunocytokine (interleukin-2 variant; IgG-IL2v) and tumor- or fibroblast-targeted T cell bispecific antibody (TCB). Following treatment with IgG-IL2v, activation of T cells, NK cells, and NKT cells was demonstrated by increased expression of the activation marker CD69 and enhanced cytokine secretion. The combination of TCBs with IgG-IL2v molecules was more effective than monotherapy, as shown by enhanced effects on immune cell infiltration; activation; increased cytokine secretion; and faster, more efficient elimination of targeted cells. This study demonstrates that the 3D heterotypic spheroid model provides a novel and versatile tool for in vitro evaluation of cancer immunotherapy agents and allows for assessment of additional aspects of the activity of cancer immunotherapy agents, including analysis of immune cell infiltration and drug targeting.

In Vivo Fluorescence Imaging of the Activity of CEA TCB, a Novel T-Cell Bispecific Antibody, Reveals Highly Specific Tumor Targeting and Fast Induction of T-Cell-Mediated Tumor Killing.

PURPOSE: CEA TCB (RG7802, RO6958688) is a novel T-cell bispecific antibody, engaging CD3ε upon binding to carcinoembryonic antigen (CEA) on tumor cells. Containing an engineered Fc region, conferring an extended blood half-life while preventing side effects due to activation of innate effector cells, CEA TCB potently induces tumor lysis in mouse tumors. Here we aimed to characterize the pharmacokinetic profile, the biodistribution, and the mode of action of CEA TCB by combining in vitro and in vivo fluorescence imaging readouts. EXPERIMENTAL DESIGN: CEA-expressing tumor cells (LS174T) and human peripheral blood mononuclear cells (PBMC) were cocultured in vitro or cografted into immunocompromised mice. Fluorescence reflectance imaging and intravital 2-photon (2P) microscopy were employed to analyze in vivo tumor targeting while in vitro confocal and intravital time-lapse imaging were used to assess the mode of action of CEA TCB. RESULTS: Fluorescence reflectance imaging revealed increased ratios of extravascular to vascular fluorescence signals in tumors after treatment with CEA TCB compared with control antibody, suggesting specific targeting, which was confirmed by intravital microscopy. Confocal and intravital 2P microscopy showed CEA TCB to accelerate T-cell-dependent tumor cell lysis by inducing a local increase of effector to tumor cell ratios and stable crosslinking of multiple T cells to individual tumor cells. CONCLUSIONS: Using optical imaging, we demonstrate specific tumor targeting and characterize the mode of CEA TCB-mediated target cell lysis in a mouse tumor model, which supports further clinical evaluation of CEA TCB. Clin Cancer Res; 22(17); 4417-27. ©2016 AACRSee related commentary by Teijeira et al., p. 4277.

A Novel Carcinoembryonic Antigen T-Cell Bispecific Antibody (CEA TCB) for the Treatment of Solid Tumors.

PURPOSE: CEA TCB is a novel IgG-based T-cell bispecific (TCB) antibody for the treatment of CEA-expressing solid tumors currently in phase I clinical trials (NCT02324257). Its format incorporates bivalent binding to CEA, a head-to-tail fusion of CEA- and CD3e-binding Fab domains and an engineered Fc region with completely abolished binding to FcγRs and C1q. The study provides novel mechanistic insights into the activity and mode of action of CEA TCB. EXPERIMENTAL DESIGN: CEA TCB activity was characterized on 110 cell lines in vitro and in xenograft tumor models in vivo using NOG mice engrafted with human peripheral blood mononuclear cells. RESULTS: Simultaneous binding of CEA TCB to tumor and T cells leads to formation of immunologic synapses, T-cell activation, secretion of cytotoxic granules, and tumor cell lysis. CEA TCB activity strongly correlates with CEA expression, with higher potency observed in highly CEA-expressing tumor cells and a threshold of approximately 10,000 CEA-binding sites/cell, which allows distinguishing between high- and low-CEA-expressing tumor and primary epithelial cells, respectively. Genetic factors do not affect CEA TCB activity confirming that CEA expression level is the strongest predictor of CEA TCB activity. In vivo, CEA TCB induces regression of CEA-expressing xenograft tumors with variable amounts of immune cell infiltrate, leads to increased frequency of activated T cells, and converts PD-L1 negative into PD-L1-positive tumors. CONCLUSIONS: CEA TCB is a novel generation TCB displaying potent antitumor activity; it is efficacious in poorly infiltrated tumors where it increases T-cell infiltration and generates a highly inflamed tumor microenvironment. Clin Cancer Res; 22(13); 3286-97. ©2016 AACR.

¹¹¹In-anti-F4/80-A3-1 antibody: a novel tracer to image macrophages.

PURPOSE: Here, the expression of F4/80 on the cell surface of murine macrophages was exploited to develop a novel imaging tracer that could visualize macrophages in vivo. METHODS: The immunoreactive fraction and IC50 of anti-F4/80-A3-1, conjugated with diethylenetriaminepentaacetic acid (DTPA) and radiolabelled with (111)In, were determined in vitro using murine bone marrow-derived macrophages. In vivo biodistribution studies were performed with (111)In-anti-F4/80-A3-1 and isotype-matched control antibody (111)In-rat IgG2b at 24 and 72 h post-injection (p.i.) in SCID/Beige mice bearing orthotopic MDA-MB-231 xenografts. In some studies mice were also treated with liposomal clodronate. Macrophage content in tissues was determined immunohistochemically. Micro-single photon emission computed tomography (SPECT)/CT images were also acquired. RESULTS: In vitro binding assays showed that (111)In-anti-F4/80-A3-1 specifically binds F4/80 receptor-positive macrophages. The immunoreactivity of anti-F4/80-A3-1 was 75 % and IC50 was 0.58 nM. In vivo, injection of 10 or 100 µg (111)In-anti-F4/80-A3-1 resulted in splenic uptake of 78 %ID/g and 31 %ID/g, respectively, and tumour uptake of 1.38 %ID/g and 4.08 %ID/g, respectively (72 h p.i.). Liposomal clodronate treatment reduced splenic uptake of 10 µg (111)In-anti-F4/80-A3-1 from 248 %ID/g to 114 %ID/g and reduced (111)In-anti-F4/80-A3-1 uptake in the liver and femur (24 h p.i.). Tracer retention in the blood and tumour uptake increased (24 h p.i.). Tumour uptake of (111)In-anti-F4/80-A3-1 was visualized by microSPECT/CT. Macrophage density in the spleen and liver decreased in mice treated with liposomal clodronate. Uptake of (111)In-rat IgG2b was lower in the spleen, liver and femur when compared to (111)In-anti-F4/80-A3-1. CONCLUSION: Radiolabelled anti-F4/80-A3-1 antibodies specifically localize in tissues infiltrated by macrophages in mice and can be used to visualize tumours. The liver and spleen act as antigen sink organs for macrophage-specific tracers.

Self-reported neck pain is associated with migraine but not with tension-type headache in adolescents.

AIM: The aim of the present analysis is to confirm or refute the association of neck pain to migraine or tension-type headache and to assess whether this association is independent of other risk factors for headache. METHODS: Secondary school students were invited to complete a questionnaire on headache and lifestyle factors in a cross-sectional study. Neck pain was assessed via (a) a screening question concerning neck pain and (b) denoting affected areas in schematic drawings of the human body. RESULTS: Absolute increment in prevalence of headache with pain in the shoulder-neck region was between 7.5% and 9.6%. Gender, grade, stress and lifestyle factors were assessed as potential confounding factors. Nearly all factors were associated with shoulder-neck pain and most with headache. After adjustment for confounders, the association of neck pain with headache was almost completely confined to migraine (OR 2.39; 95% CI 1.48-3.85) and migraine + tension-type headache (OR 2.12; 95% CI 1.50-2.99), whereas the association with isolated tension-type headache was negligible (OR 1.22, 95% CI 0.87-1.69). CONCLUSION: Neck pain is associated with migraine but not with tension-type headache. A possible link between migraine and neck pain may be the cervico-trigeminal convergence of neck and meningeal sensory afferents or a disturbed descending inhibition in migraine.

How specific are risk factors for headache in adolescents? Results from a cross-sectional study in Germany.

BACKGROUND: The identified preventable risk factors for primary headache in adolescents are smoking; consumption of coffee or alcoholic mixed drinks; physical inactivity; muscle pain in the head, neck, or shoulder region; and chronic stress. OBJECTIVE: To investigate the interrelation of headache with other health complaints and the specificity of the above-mentioned risk factors for headache in adolescents. METHODS: A total of 1,260 students (grades 10 and 11) filled in questionnaires on headache, dietary, and lifestyle factors. The type of headache and health complaints such as dizziness, abdominal pain, musculoskeletal pains, symptoms of possible fatigue syndrome, and psychic complaints were assessed. RESULTS: Isolated headache was found in 18% of the headache sufferers; most frequently isolated tension-type headache (78.2%). Only among adolescents with a combination of headache (mainly migraine) and other health complaints, significant associations for almost all analyzed risk factors were found. The strength of the associations with the considered risk factors was very similar in all three analyzed strata except for considerably lower odds ratios for isolated headache. CONCLUSION: All analyzed risk factors are nonspecific for headache in adolescents because they also increase the risk for other health complaints. Interventions, therefore, should consider a holistic approach focusing not only on headache but also on a broader spectrum of health complaints.

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response.

INTRODUCTION: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined. METHODS: A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion. RESULTS: Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids. CONCLUSIONS: Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

Pattern analysis accounts for heterogeneity observed in MRI studies of tumor angiogenesis.

MRI is a method of choice for assessing anatomical structures or angiogenesis-related parameters noninvasively during tumor progression. Typically, tumor tissue displays a high degree of heterogeneity that can be evaluated using pattern analysis (PA), which comprises shape and texture analysis. This work aims at implementing PA methods to study angiogenesis in a murine tumor model and testing its sensitivity with regard to detecting changes elicited by administration of a drug. Twelve balb/c-nude mice were injected subcutaneously with 10(6) C51 cells (colon carcinoma). A first group (N = 6) of animals was treated with dimethyloxalylglycine, a drug known to stabilize hypoxia-inducible-factor-α, which among other functions, is involved in angiogenesis. The second group (N = 6) was treated with saline. MRI experiments assessing tumor blood volume and permeability-maps (K(trans) ) were performed immediately before and 6 days after drug treatment. Data have been analyzed using standard histogram analysis and PA. Standard histogram analysis did not reveal any difference between the two groups, neither before nor after the treatment. In contrast, PA revealed significant differences between drug and placebo treated mice in the texture of the TBV and K(trans) maps after drug treatment, but not with regard to tumors shapes. The results indicated that in view of the heterogeneity of tumor tissue, standard histogram analysis appears insensitive in picking-up differences in response to treatment, while PA appears to be particularly sensitive to changes in texture.

Prevention of headache in adolescents: population-attributable risk fraction for risk factors amenable to intervention.

INTRODUCTION: Several risk factors for headache have been identified, some of which are potentially amenable to interventions. The potential effect of such interventions can be predicted by the population-attributable risk fraction (PARF). We assessed PARFs of the the following risk factors: neck muscle pain, chronic stress, alcohol consumption, smoking, coffee consumption, and physical inactivity. We studied the maximal possible effect achievable by avoidance of these risk factors. METHODS: Two approaches to estimate PARFs are compared, which assess their cumulative and individual impact of risk factors by age: the Levin formula and the average attributable fraction. RESULTS: The overall impact for removal of all six risk factors amounts to 19.7% for the average attributable fraction. Neck tension and consumption of alcohol ranked as the strongest population-attributable risk factor for any headache. The potential impact for migraine was considerably higher (43.8%). With increasing age, the overall impact of risk factors on headache increases by 18.9%. CONCLUSION: Based on the estimations of the most appropriate approach, up to 20% of headaches in general and up to 43% of migraine in adolescents might be preventable by removing risk factors amenable to intervention, with increasing proportions by age.

Recording intracellular molecular events from the outside: glycosylphosphatidylinositol-anchored avidin as a reporter protein for in vivo imaging.

UNLABELLED: With the emergence of multimodal imaging strategies, genetically encoded reporters that can be flexibly combined with any imaging modality become highly attractive. Here we describe the use of glycosylphosphatidylinositol (GPI)-anchored avidin, an avidin moiety targeted to the extracellular side of cell membranes via a GPI anchor, as a reporter for in vivo imaging. Being present on the outside of cells, avidin can be visualized with any type of biotinylated imaging agent, without the requirement that the probe be membrane-permeable. We used the avidin-GPI system to monitor the activity of hypoxia-inducible factors (HIFs)-oxygen-sensing transcription factors, which play a major role in regulating cancer progression-in a mouse tumor allograft model. METHODS: Mouse C51 cells were stably transfected with pH3SVG, a reporter construct driving the expression of avidin-GPI from an HIF-sensitive promoter. The transfected cells were subcutaneously implanted into BALB/c nude mice. At 10 d after tumor inoculation, mice received an intravenous injection of either alexa-594-biocytin or (67)Ga-DOTA-biotin, and tumor HIF activity was imaged using fluorescence reflectance imaging or SPECT. RESULTS: In vitro cell experiments demonstrated the functionality and HIF-dependent regulation of the avidin-GPI reporter construct. In vivo, avidin-GPI was targeted specifically in allograft tumors with biotinylated imaging probes using both fluorescence imaging and SPECT. Analysis of the reporter expression pattern on ex vivo tumor tissue sections indicated a good overlap, with areas of hypoxia. CONCLUSION: We have demonstrated the utility of avidin-GPI as a reporter for multimodal in vivo imaging using both a fluorescence and a SPECT approach to assess intracellular oxygen signaling in a mouse tumor model.

Longitudinal and multimodal in vivo imaging of tumor hypoxia and its downstream molecular events.

Tumor hypoxia and the hypoxia-inducible factors (HIFs) play a central role in the development of cancer. To study the relationship between tumor growth, tumor hypoxia, the stabilization of HIF-1alpha, and HIF transcriptional activity, we have established an in vivo imaging tool that allows longitudinal and noninvasive monitoring of these processes in a mouse C51 allograft tumor model. We used positron emission tomography (PET) with the hypoxia-sensitive tracer [(18)F]-fluoromisonidazole (FMISO) to measure tumor hypoxia over 14 days. Stabilization of HIF-1alpha and HIF transcriptional activity were assessed by bioluminescence imaging using the reporter constructs HIF-1alpha-luciferase and hypoxia response element-luciferase, respectively, stably expressed in C51 cells. Interestingly, we did not observe any major change in the level of tumor hypoxia throughout the observation period whereas HIF-1alpha levels and HIF activity showed drastic temporal variations. When comparing the readouts as a function of time we found a good correlation between HIF-1alpha levels and HIF activity. In contrast, there was no significant correlation between the [(18)F]-FMISO PET and HIF readouts. The tool developed in this work allows for the longitudinal study of tumor hypoxia and HIF-1alpha in cancer in an individual animal and will be of value when monitoring the efficacy of therapeutical interventions targeting the HIF pathway.