A bispecific Clec9A-PD-L1 targeted type I interferon profoundly reshapes the tumor microenvironment towards an antitumor state.

Despite major improvements in immunotherapeutic strategies, the immunosuppressive tumor microenvironment remains a major obstacle for the induction of efficient antitumor responses. In this study, we show that local delivery of a bispecific Clec9A-PD-L1 targeted type I interferon (AcTaferon, AFN) overcomes this hurdle by reshaping the tumor immune landscape.Treatment with the bispecific AFN resulted in the presence of pro-immunogenic tumor-associated macrophages and neutrophils, increased motility and maturation profile of cDC1 and presence of inflammatory cDC2. Moreover, we report empowered diversity in the CD8+ T cell repertoire and induction of a shift from naive, dysfunctional CD8+ T cells towards effector, plastic cytotoxic T lymphocytes together with increased presence of NK and NKT cells as well as decreased regulatory T cell levels. These dynamic changes were associated with potent antitumor activity. Tumor clearance and immunological memory, therapeutic immunity on large established tumors and blunted tumor growth at distant sites were obtained upon co-administration of a non-curative dose of chemotherapy.Overall, this study illuminates further application of type I interferon as a safe and efficient way to reshape the suppressive tumor microenvironment and induce potent antitumor immunity; features which are of major importance in overcoming the development of metastases and tumor cell resistance to immune attack. The strategy described here has potential for application across to a broad range of cancer types.

Selective IL-1 activity on CD8+ T cells empowers antitumor immunity and synergizes with neovasculature-targeted TNF for full tumor eradication.

BACKGROUND: Clinical success of therapeutic cancer vaccines depends on the ability to mount strong and durable antitumor T cell responses. To achieve this, potent cellular adjuvants are highly needed. Interleukin-1ß (IL-1ß) acts on CD8+ T cells and promotes their expansion and effector differentiation, but toxicity and undesired tumor-promoting side effects hamper efficient clinical application of this cytokine. METHODS: This 'cytokine problem' can be solved by use of AcTakines (Activity-on-Target cytokines), which represent fusions between low-activity cytokine mutants and cell type-specific single-domain antibodies. AcTakines deliver cytokine activity to a priori selected cell types and as such evade toxicity and unwanted off-target side effects. Here, we employ subcutaneous melanoma and lung carcinoma models to evaluate the antitumor effects of AcTakines. RESULTS: In this work, we use an IL-1ß-based AcTakine to drive proliferation and effector functionality of antitumor CD8+ T cells without inducing measurable toxicity. AcTakine treatment enhances diversity of the T cell receptor repertoire and empowers adoptive T cell transfer. Combination treatment with a neovasculature-targeted tumor necrosis factor (TNF) AcTakine mediates full tumor eradication and establishes immunological memory that protects against secondary tumor challenge. Interferon-γ was found to empower this AcTakine synergy by sensitizing the tumor microenvironment to TNF. CONCLUSIONS: Our data illustrate that anticancer cellular immunity can be safely promoted with an IL-1ß-based AcTakine, which synergizes with other immunotherapies for efficient tumor destruction.

Microcirculatory Function during Endotoxemia-A Functional Citrulline-Arginine-NO Pathway and NOS3 Complex Is Essential to Maintain the Microcirculation.

Competition for the amino acid arginine by endothelial nitric-oxide synthase (NOS3) and (pro-)inflammatory NO-synthase (NOS2) during endotoxemia appears essential in the derangement of the microcirculatory flow. This study investigated the role of NOS2 and NOS3 combined with/without citrulline supplementation on the NO-production and microcirculation during endotoxemia. Wildtype (C57BL6/N background; control; n = 36), Nos2-deficient, (n = 40), Nos3-deficient (n = 39) and Nos2/Nos3-deficient mice (n = 42) received a continuous intravenous LPS infusion alone (200 µg total, 18 h) or combined with L-citrulline (37.5 mg, last 6 h). The intestinal microcirculatory flow was measured by side-stream dark field (SDF)-imaging. The jejunal intracellular NO production was quantified by in vivo NO-spin trapping combined with electron spin-resonance (ESR) spectrometry. Amino-acid concentrations were measured by high-performance liquid chromatography (HPLC). LPS infusion decreased plasma arginine concentration in control and Nos3-/- compared to Nos2-/- mice. Jejunal NO production and the microcirculation were significantly decreased in control and Nos2-/- mice after LPS infusion. No beneficial effects of L-citrulline supplementation on microcirculatory flow were found in Nos3-/- or Nos2-/-/Nos3-/- mice. This study confirms that L-citrulline supplementation enhances de novo arginine synthesis and NO production in mice during endotoxemia with a functional NOS3-enzyme (control and Nos2-/- mice), as this beneficial effect was absent in Nos3-/- or Nos2-/-/Nos3-/- mice.

Targeting IFN activity to both B cells and plasmacytoid dendritic cells induces a robust tolerogenic response and protection against EAE.

Type I Interferon (IFN) was the very first drug approved for the treatment of Multiple Sclerosis (MS), and is still frequently used as a first line therapy. However, systemic IFN also causes considerable side effects, affecting therapy adherence and dose escalation. In addition, the mechanism of action of IFN in MS is multifactorial and still not completely understood. Using AcTaferons (Activity-on-Target IFNs, AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting, we have previously demonstrated that specific targeting of IFN activity to dendritic cells (DCs) can protect against experimental autoimmune encephalitis (EAE), inducing in vivo tolerogenic protective effects, evidenced by increased indoleamine-2,3-dioxygenase (IDO) and transforming growth factor ß (TGFß) release by plasmacytoid (p) DCs and improved immunosuppressive capacity of regulatory T and B cells. We here report that targeting type I IFN activity specifically towards B cells also provides strong protection against EAE, and that targeting pDCs using SiglecH-AFN can significantly add to this protective effect. The superior protection achieved by simultaneous targeting of both B lymphocytes and pDCs correlated with improved IL-10 responses in B cells and conventional cDCs, and with a previously unseen very robust IDO response in several cells, including all B and T lymphocytes, cDC1 and cDC2.

Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade.

Monoclonal antibodies that target the inhibitory immune checkpoint axis consisting of programmed cell death protein 1 (PD-1) and its ligand, PD-L1, have changed the immune-oncology field. We identified K2, an anti-human PD-L1 single-domain antibody fragment, that can enhance T cell activation and tumor cell killing. In this study, the potential of different K2 formats as immune checkpoint blocking medicines was evaluated using a gene-based delivery approach. We showed that 2K2 and 3K2, a bivalent and trivalent K2 format generated using a 12 GS (glycine-serine) linker, were 313- and 135-fold more potent in enhancing T cell receptor (TCR) signaling in PD-1POS cells than was monovalent K2. We further showed that bivalent constructs generated using a 30 GS linker or disulfide bond were 169- and 35-fold less potent in enhancing TCR signaling than was 2K2. 2K2 enhanced tumor cell killing in a 3D melanoma model, albeit to a lesser extent than avelumab. Therefore, an immunoglobulin (Ig)G1 antibody-like fusion protein was generated, referred to as K2-Fc. K2-Fc was significantly better than avelumab in enhancing tumor cell killing in the 3D melanoma model. Overall, this study describes K2-based immune checkpoint medicines, and it highlights the benefit of an IgG1 Fc fusion to K2 that gains bivalency, effector functions, and efficacy.

Tolerizing Strategies for the Treatment of Autoimmune Diseases: From ex vivo to in vivo Strategies.

Autoimmune diseases such as multiple sclerosis (MS), type I diabetes (T1D), inflammatory bowel diseases (IBD), and rheumatoid arthritis (RA) are chronic, incurable, incapacitating and at times even lethal conditions. Worldwide, millions of people are affected, predominantly women, and their number is steadily increasing. Currently, autoimmune patients require lifelong immunosuppressive therapy, often accompanied by severe adverse side effects and risks. Targeting the fundamental cause of autoimmunity, which is the loss of tolerance to self- or innocuous antigens, may be achieved via various mechanisms. Recently, tolerance-inducing cellular therapies, such as tolerogenic dendritic cells (tolDCs) and regulatory T cells (Tregs), have gained considerable interest. Their safety has already been evaluated in patients with MS, arthritis, T1D, and Crohn's disease, and clinical trials are underway to confirm their safety and therapeutic potential. Cell-based therapies are inevitably expensive and time-consuming, requiring laborious ex vivo manufacturing. Therefore, direct in vivo targeting of tolerogenic cell types offers an attractive alternative, and several strategies are being explored. Type I IFN was the first disease-modifying therapy approved for MS patients, and approaches to endogenously induce IFN in autoimmune diseases are being pursued vigorously. We here review and discuss tolerogenic cellular therapies and targeted in vivo tolerance approaches and propose a novel strategy for cell-specific delivery of type I IFN signaling to a cell type of choice.

Safe eradication of large established tumors using neovasculature-targeted tumor necrosis factor-based therapies.

Systemic toxicities have severely limited the clinical application of tumor necrosis factor (TNF) as an anticancer agent. Activity-on-Target cytokines (AcTakines) are a novel class of immunocytokines with improved therapeutic index. A TNF-based AcTakine targeted to CD13 enables selective activation of the tumor neovasculature without any detectable toxicity in vivo. Upregulation of adhesion markers supports enhanced T-cell infiltration leading to control or elimination of solid tumors by, respectively, CAR T cells or a combination therapy with CD8-targeted type I interferon AcTakine. Co-treatment with a CD13-targeted type II interferon AcTakine leads to very rapid destruction of the tumor neovasculature and complete regression of large, established tumors. As no tumor markers are needed, safe and efficacious elimination of a broad range of tumor types becomes feasible.

Targeting interferon activity to dendritic cells enables in vivo tolerization and protection against EAE in mice.

Type I Interferon (IFN) is widely used for multiple sclerosis (MS) treatment, but its side effects are limiting and its mechanism of action still unknown. Furthermore, 30-50% of MS patients are unresponsive, and IFN can even induce relapses. Fundamental understanding of the cellular target(s) of IFN will help to optimize treatments by reducing side effects and separating beneficial from detrimental effects. To improve clinical systemic IFN usage, we are developing AcTaferons (Activity-on-Target IFNs = AFNs), optimized IFN-based immunocytokines that allow cell-specific targeting. In experimental autoimmune encephalitis (EAE) in mice, high dose WT mIFNα could delay disease, but caused mortality and severe hematological deficits. In contrast, AFN targeted to dendritic cells (DC, via Clec9A) protected without mortality or hematological consequences. Conversely, CD8-targeted AFN did not protect and exacerbated weight loss, indicating the presence of both protective and unfavorable IFN effects in EAE. Comparing Clec9A-, XCR1-and SiglecH-targeting, we found that targeting AFN to plasmacytoid (p) and conventional (c) DC is superior and non-toxic compared to WT mIFN. DC-targeted AFN increased pDC numbers and their tolerogenic potential, evidenced by increased TGFß and IDO synthesis and regulatory T cell induction. In addition, both regulatory T and B cells produced significantly more immunosuppressive TGFß and IL-10. In conclusion, specific DC-targeting of IFN activity induces a robust in vivo tolerization, efficiently protecting against EAE, without noticeable side effects. Thus, dissecting positive and negative IFN effects via cell-specific targeting may result in better and safer MS therapy and response rates.

A safe and highly efficient tumor-targeted type I interferon immunotherapy depends on the tumor microenvironment.

Despite approval for the treatment of various malignancies, clinical application of cytokines such as type I interferon (IFN) is severely impeded by their systemic toxicity. AcTakines (Activity-on-Target cytokines) are optimized immunocytokines that, when injected in mice, only reveal their activity upon cell-specific impact. We here show that type I IFN-derived AcTaferon targeted to the tumor displays strong antitumor activity without any associated toxicity, in contrast with wild type IFN. Treatment with CD20-targeted AcTaferon of CD20+ lymphoma tumors or melanoma tumors engineered to be CD20+, drastically reduced tumor growth. This antitumor effect was completely lost in IFNAR- or Batf3-deficient mice, and depended on IFN signaling in conventional dendritic cells. Also the presence of, but not the IFN signaling in, CD8+ T lymphocytes was critical for proficient antitumor effects. When combined with immunogenic chemotherapy, low-dose TNF, or immune checkpoint blockade strategies such as anti-PDL1, anti-CTLA4 or anti-LAG3, complete tumor regressions and subsequent immunity (memory) were observed, still without any concomitant morbidity, again in sharp contrast with wild type IFN. Interestingly, the combination therapy of tumor-targeted AcTaferon with checkpoint inhibiting antibodies indicated its ability to convert nonresponding tumors into responders. Collectively, our findings demonstrate that AcTaferon targeted to tumor-specific surface markers may provide a safe and generic addition to cancer (immuno)therapies.

Delivering Type I Interferon to Dendritic Cells Empowers Tumor Eradication and Immune Combination Treatments.

An ideal generic cancer immunotherapy should mobilize the immune system to destroy tumor cells without harming healthy cells and remain active in case of recurrence. Furthermore, it should preferably not rely on tumor-specific surface markers, as these are only available in a limited set of malignancies. Despite approval for treatment of various cancers, clinical application of cytokines is still impeded by their multiple toxic side effects. Type I IFN has a long history in the treatment of cancer, but its multifaceted activity pattern and complex side effects prevent its clinical use. Here we develop AcTakines (Activity-on-Target cytokines), optimized (mutated) immunocytokines that are up to 1,000-fold more potent on target cells, allowing specific signaling in selected cell types only. Type I IFN-derived AcTaferon (AFN)-targeting Clec9A+ dendritic cells (DC) displayed strong antitumor activity in murine melanoma, breast carcinoma, and lymphoma models and against human lymphoma in humanized mice without any detectable toxic side effects. Combined with immune checkpoint blockade, chemotherapy, or low-dose TNF, complete tumor regression and long-lasting tumor immunity were observed, still without adverse effects. Our findings indicate that DC-targeted AFNs provide a novel class of highly efficient, safe, and broad-spectrum off-the-shelf cancer immunotherapeutics with no need for a tumor marker.Significance: Targeted type I interferon elicits powerful antitumor efficacy, similar to wild-type IFN, but without any toxic side effects. Cancer Res; 78(2); 463-74. ©2017 AACR.

Cardiovascular and pharmacological implications of haem-deficient NO-unresponsive soluble guanylate cyclase knock-in mice.

Oxidative stress, a central mediator of cardiovascular disease, results in loss of the prosthetic haem group of soluble guanylate cyclase (sGC), preventing its activation by nitric oxide (NO). Here we introduce Apo-sGC mice expressing haem-free sGC. Apo-sGC mice are viable and develop hypertension. The haemodynamic effects of NO are abolished, but those of the sGC activator cinaciguat are enhanced in apo-sGC mice, suggesting that the effects of NO on smooth muscle relaxation, blood pressure regulation and inhibition of platelet aggregation require sGC activation by NO. Tumour necrosis factor (TNF)-induced hypotension and mortality are preserved in apo-sGC mice, indicating that pathways other than sGC signalling mediate the cardiovascular collapse in shock. Apo-sGC mice allow for differentiation between sGC-dependent and -independent NO effects and between haem-dependent and -independent sGC effects. Apo-sGC mice represent a unique experimental platform to study the in vivo consequences of sGC oxidation and the therapeutic potential of sGC activators.

Citrulline Supplementation Improves Organ Perfusion and Arginine Availability under Conditions with Enhanced Arginase Activity.

Enhanced arginase-induced arginine consumption is believed to play a key role in the pathogenesis of sickle cell disease-induced end organ failure. Enhancement of arginine availability with L-arginine supplementation exhibited less consistent results; however, L-citrulline, the precursor of L-arginine, may be a promising alternative. In this study, we determined the effects of L-citrulline compared to L-arginine supplementation on arginine-nitric oxide (NO) metabolism, arginine availability and microcirculation in a murine model with acutely-enhanced arginase activity. The effects were measured in six groups of mice (n = 8 each) injected intraperitoneally with sterile saline or arginase (1000 IE/mouse) with or without being separately injected with L-citrulline or L-arginine 1 h prior to assessment of the microcirculation with side stream dark-field (SDF)-imaging or in vivo NO-production with electron spin resonance (ESR) spectroscopy. Arginase injection caused a decrease in plasma and tissue arginine concentrations. L-arginine and L-citrulline supplementation both enhanced plasma and tissue arginine concentrations in arginase-injected mice. However, only the citrulline supplementation increased NO production and improved microcirculatory flow in arginase-injected mice. In conclusion, the present study provides for the first time in vivo experimental evidence that L-citrulline, and not L-arginine supplementation, improves the end organ microcirculation during conditions with acute arginase-induced arginine deficiency by increasing the NO concentration in tissues.

MAPK-activated protein kinase 2-deficiency causes hyperacute tumor necrosis factor-induced inflammatory shock.

BACKGROUND: MAPK-activated protein kinase 2 (MK2) plays a pivotal role in the cell response to (inflammatory) stress. Among others, MK2 is known to be involved in the regulation of cytokine mRNA metabolism and regulation of actin cytoskeleton dynamics. Previously, MK2-deficient mice were shown to be highly resistant to LPS/d-Galactosamine-induced hepatitis. Additionally, research in various disease models has indicated the kinase as an interesting inhibitory drug target for various acute or chronic inflammatory diseases. RESULTS: We show that in striking contrast to the known resistance of MK2-deficient mice to a challenge with LPS/D-Gal, a low dose of tumor necrosis factor (TNF) causes hyperacute mortality via an oxidative stress driven mechanism. We identified in vivo defects in the stress fiber response in endothelial cells, which could have resulted in reduced resistance of the endothelial barrier to deal with exposure to oxidative stress. In addition, MK2-deficient mice were found to be more sensitive to cecal ligation and puncture-induced sepsis. CONCLUSIONS: The capacity of the endothelial barrier to deal with inflammatory and oxidative stress is imperative to allow a regulated immune response and maintain endothelial barrier integrity. Our results indicate that, considering the central role of TNF in pro-inflammatory signaling, therapeutic strategies examining pharmacological inhibition of MK2 should take potentially dangerous side effects at the level of endothelial barrier integrity into account.

A multiscale entropy-based tool for scoring severity of systemic inflammation.

OBJECTIVE: Early detection and start of appropriate treatment are highly correlated with survival of sepsis and septic shock, but the currently available predictive tools are not sensitive enough to identify patients at risk. DESIGN: Linear (time and frequency domain) and nonlinear (unifractal and multiscale complexity dynamics) measures of beat-to-beat interval variability were analyzed in two mouse models of inflammatory shock to determine if they are sensitive enough to predict outcome. SETTING: University research laboratory. SUBJECTS: Blood pressure transmitter-implanted female C57BL/6J mice. INTERVENTIONS: IV administration of tumor necrosis factor (n = 11) or lipopolysaccharide (n = 14). MEASUREMENTS AND MAIN RESULTS: Contrary to linear indices of variability, unifractal dynamics, and absolute heart rate or blood pressure, quantification of complex beat-to-beat dynamics using multiscale entropy was able to predict survival outcome starting as early as 40 minutes after induction of inflammatory shock. Based on these results, a new and clinically relevant index of multiscale entropy was developed that scores the key features of a multiscale entropy profile. Contrary to multiscale entropy, multiscale entropy scoring can be followed as a function of time to monitor disease progression with limited loss of information. CONCLUSIONS: Analysis of multiscale complexity of beat-to-beat dynamics at high temporal resolution has potential as a sensitive prognostic tool with translational power that can predict survival outcome in systemic inflammatory conditions such as sepsis and septic shock.

Simultaneous targeting of IL-1 and IL-18 is required for protection against inflammatory and septic shock.

RATIONALE: Sepsis is one of the leading causes of death around the world. The failure of clinical trials to treat sepsis demonstrates that the molecular mechanisms are multiple and are still insufficiently understood. OBJECTIVES: To clarify the long disputed hierarchical contribution of several central inflammatory mediators (IL-1ß, IL-18, caspase [CASP] 7, CASP1, and CASP11) in septic shock and to explore their therapeutic potential. METHODS: LPS- and tumor necrosis factor (TNF)-induced lethal shock, and cecal ligation and puncture (CLP) were performed in genetically or pharmacologically targeted mice. Body temperature and survival were monitored closely, and plasma was analyzed for several markers of cellular disintegration and inflammation. MEASUREMENTS AND MAIN RESULTS: Interestingly, deficiency of both IL-1ß and IL-18 additively prevented LPS-induced mortality. The detrimental role of IL-1ß and IL-18 was confirmed in mice subjected to a lethal dose of TNF, or to a lethal CLP procedure. Although their upstream activator, CASP1, and its amplifier, CASP11, are considered potential therapeutic targets because of their crucial involvement in endotoxin-induced toxicity, CASP11- or CASP1/11-deficient mice were not, or hardly, protected against a lethal TNF or CLP challenge. In line with our results obtained in genetically deficient mice, only the combined neutralization of IL-1 and IL-18, using the IL-1 receptor antagonist anakinra and anti-IL-18 antibodies, conferred complete protection against endotoxin-induced lethality. CONCLUSIONS: Our data point toward the therapeutic potential of neutralizing IL-1 and IL-18 simultaneously in sepsis, rather than inhibiting the upstream inflammatory caspases.

Nitric oxide production by endotoxin preparations in TLR4-deficient mice.

Sepsis and septic shock result from an exacerbated systemic inflammatory reaction to infection. Their incidence is rising, and they have recently become the main cause of death in intensive care units. Septic shock is defined as sepsis accompanied by life-threatening refractory hypotension, for which excessive nitric oxide (NO), produced by inducible NO synthase iNOS, is thought responsible. LPS, a vital outer membrane component of Gram-negative bacteria, mimics most of the septic effects and is widely used as a model for septic shock. TLR4 is the signal-transducing receptor for LPS, evidenced by the resistance of TLR4-deficient C3H/HeJ and C57BL/10ScNJ mice. As expected, we found that TLR4 deficiency precludes LPS-induced cytokine production, independent of the purity of the LPS preparation. However, various conventional LPS preparations induced NO in TLR4-deficient mice to the same level as in control animals, while ultrapure LPS did not, indicating the presence of NO-producing contaminant(s). Nevertheless, despite identical iNOS induction pattern and systemic NO levels, the contaminant does not cause hypotension, hypothermia, or any other sign of morbidity. Using mice deficient for TLR2, TRL3, TLR4, TRL2x4, TLR9, MyD88 or TRIF, we found that the contaminant signals via TLR2 and MyD88. In conclusion, conventional LPS preparations generally used in endotoxic shock research contain TLR2 agonists that induce iNOS and high levels of systemic NO as such, and synergize with LPS towards the production of pro-inflammatory cytokines, morbidity and mortality. Surprisingly, the excessive iNOS-derived systemic NO production induced by impure LPS in TLR4⁻/⁻ is not accompanied by hypotension or morbidity.

The soluble guanylate cyclase activator BAY 58-2667 protects against morbidity and mortality in endotoxic shock by recoupling organ systems.

Sepsis and septic shock are associated with high mortality rates and the majority of sepsis patients die due to complications of multiple organ failure (MOF). The cyclic GMP (cGMP) producing enzyme soluble guanylate cyclase (sGC) is crucially involved in the regulation of (micro)vascular homeostasis, cardiac function and, consequently, organ function. However, it can become inactivated when exposed to reactive oxygen species (ROS). The resulting heme-free sGC can be reactivated by the heme- and nitric oxide (NO)-independent sGC activator BAY 58-2667 (Cinaciguat). We report that late (+8 h) post-treatment with BAY 58-2667 in a mouse model can protect against lethal endotoxic shock. Protection was associated with reduced hypothermia, circulating IL-6 levels, cardiomyocyte apoptosis, and mortality. In contrast to BAY 58-2667, the sGC stimulator BAY 41-2272 and the phosphodiesterase 5 inhibitor Sildenafil did not have any beneficial effect on survival, emphasizing the importance of the selectivity of BAY 58-2667 for diseased vessels and tissues. Hemodynamic parameters (blood pressure and heart rate) were decreased, and linear and nonlinear indices of blood pressure variability, reflective for (un)coupling of the communication between the autonomic nervous system and the heart, were improved after late protective treatment with BAY 58-2667. In conclusion, our results demonstrate the pivotal role of the NO/sGC axis in endotoxic shock. Stabilization of sGC function with BAY 58-2667 can prevent mortality when given in the correct treatment window, which probably depends on the dynamics of the heme-free sGC pool, in turn influenced by oxidative stress. We speculate that, considering the central role of sGC signaling in many pathways required for maintenance of (micro)circulatory homeostasis, BAY 58-2667 supports organ function by recoupling inter-organ communication pathways.