Expanding the armamentarium for the spondyloarthropathies

Ankylosing spondylitis (AS) is a member of the family of spondyloarthropathies, which are inflammatory arthritides largely involving the axial skeleton and commonly accompanied by peripheral arthritis. Genetic factors, particularly the presence of HLA-B27, are major contributors to the susceptibility for AS. Despite some therapeutic advances, the treatment options for patients with AS and related disorders have been limited. Several lines of evidence have led to the hypothesis that patients with AS might benefit from treatment with tumor necrosis factor (TNF). Specifically, TNF concentrations are known to be significantly elevated in the synovium of patients with rheumatoid arthritis (RA), in the inflamed gut of patients with inflammatory bowel disease, and in the inflamed sacroiliac joints of patients with AS. The anti-TNF agents have been shown to be of benefit in, and currently have indications for, RA (etanercept, infliximab, adalimumab), Crohn's disease (infliximab), and psoriatic arthritis (etanercept). Because the spondyloarthropathies share pathogenetic mechanisms with the above-specified disease states, studies have been conducted to evaluate the effectiveness of anti-TNF agents in several disorders, including AS. Data from clinical trials so far with infliximab and etanercept show that patients with AS and related disorders achieve significant improvement in clinical signs and symptoms based on validated outcomes measures. Computed tomography and magnetic resonance imaging (MRI) can facilitate the early diagnosis of AS. Studies with infliximab using MRI together with updated scoring methods demonstrated significant decreases in associated spinal inflammation. TNF antagonist therapy is well tolerated in patients with AS, with a side effect profile consistent with the prior experience of patients with RA.     

Role of tumour necrosis factor in the tumour-necrotizing activity of agents with diverging toxicity

Summary We investigated the ability of various tumournecrotizing agents with diverging toxicity to induce tumour necrosis factor (TNF) and cytostatic activity inPropionibacterium-acnes-primed Swiss and tumour-bearing BALB/c mice, and the capacity of anti-TNF antibodies to inhibit induction of tumour necrosis by the agents. Lipid A and especially its combination with muramyl dipeptide induced high TNF levels in Swiss mice, as measured in the serum. Lower levels were induced by detoxified lipid A and the nontoxic dsRNA, polyadenylic polyuridylic acid, either alone or combined with muramyl dipeptide. The toxic agents also appeared the strongest inducers of mediators with cytostatic activity against cultured endothelial cells and MethA tumour cells. Anti-TNF antibodies partially reduced the cytostatic activity of the sera against MethA cells. Tumour-bearing BALB/c mice produced only low levels of TNF and cytostatic factors in response to all agents. Recombinant mouse TNF hardly reduced the DNA synthesis of MethA cells, unless normal mouse serum was added. Serum fromP.-acnes-treated Swiss mice and tumour-bearing BALB/c mice, that were inhibitory on their own, failed to potentiate the action of TNF. Serum from Swiss mice treated with toxic, but not detoxified, lipid A caused extensive tumour necrosis upon injection into MethA-bearing BALB/c mice. This activity was completely abolished by pre-incubation of the serum with anti-TNF. The tumour-necrotizing activity of the agents could be partially reduced by prior injection of these antibodies. Results show that the capacity of the agents to induce TNF and cytostatic activity is not related to their antitumour potential. Although TNF is likely to be a crucial mediator of the tumour-necrotizing action of the toxic as well as the nontoxic agents, it is probably not the sole mediator. Data also indicate that induction of tumour necrosis does not require induction of high and, thus toxic, TNF levels in the serum.     

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.     

Simultaneous targeting of TNF and Ang2 with a novel bispecific antibody enhances efficacy in an in vivo model of arthritis

Despite the clinical success of anti-tumor necrosis factor (TNF) therapies in the treatment of inflammatory conditions such as rheumatoid arthritis, Crohn disease and psoriasis, full control of the diseases only occurs in a subset of patients and there is a need for new therapeutics with improved efficacy against broader patient populations. One possible approach is to combine biological therapeutics, but both the cost of the therapeutics and the potential for additional toxicities needs to be considered. In addition to the various mediators of immune and inflammatory pathways, angiogenesis is reported to contribute substantially to the overall pathogenesis of inflammatory diseases. The combination of an anti-angiogenic agent with anti-TNF into one molecule could be more efficacious without the risk of severe immunosuppression. To evaluate this approach with our Zybody technology, we generated bispecific antibodies that contain an Ang2 targeting peptide genetically fused to the anti-TNF antibody adalimumab (Humira®). The bispecific molecules retain the binding and functional characteristics of the anti-TNF antibody, but with additional activity that neutralizes Ang2. In a TNF transgenic mouse model of arthritis, the bispecific anti-TNF-Ang2 molecules showed a dose-dependent reduction in both clinical symptoms and histological scores that were significantly better than that achieved by adalimumab alone.     

Tumor necrosis factor, cancer and anticancer therapy

Tumor necrosis factor (TNF) is being utilized as an antineoplastic agent for the treatment of patients with locally advanced solid tumors. However, its role in cancer therapy is debated. Although a large body of evidence supports TNF's antineoplastic activity, the cascade of molecular events underlying TNF-mediated tumor regression observed in vivo is still incompletely elucidated. Intriguingly, some pre-clinical findings suggest that TNF may promote cancer development and progression, which has led to propose anti-TNF therapy as a novel approach to malignancies. In the present work, we summarize the molecular biology of TNF with particular regard to its tumor-related properties, and review the experimental and clinical evidence currently available describing the complex and sometime conflicting relationship between this cytokine, cancer and antitumor therapy. Recent insights that might pave the way to further exploitation of the antineoplastic potential of TNF are also discussed.     

TNF and TNF-receptors: From mediators of cell death and inflammation to therapeutic giants – past,present and future

Tumor Necrosis Factor (TNF), initially known for its tumor cytotoxicity, is a potent mediator of inflammation, as well as many normal physiological functions in homeostasis and health, and anti-microbial immunity. It also appears to have a central role in neurobiology, although this area of TNF biology is only recently emerging. Here, we review the basic biology of TNF and its normal effector functions, and discuss the advantages and disadvantages of therapeutic neutralization of TNF – now a commonplace practice in the treatment of a wide range of human inflammatory diseases. With over ten years of experience, and an emerging range of anti-TNF biologics now available, we also review their modes of action, which appear to be far more complex than had originally been anticipated. Finally, we highlight the current challenges for therapeutic intervention of TNF: (i) to discover and produce orally delivered small molecule TNF-inhibitors, (ii) to specifically target selected TNF producing cells or individual (diseased) tissue targets, and (iii) to pre-identify anti-TNF treatment responders. Although the future looks bright, the therapeutic modulation of TNF now moves into the era of personalized medicine with society's challenging expectations of durable treatment success and of achieving long-term disease remission.     

Differences in reactivation of tuberculosis induced from anti-TNF treatments are based on bioavailability in granulomatous tissue

The immune response to Mycobacterium tuberculosis (Mtb) infection is complex. Experimental evidence has revealed that tumor necrosis factor (TNF) plays a major role in host defense against Mtb in both active and latent phases of infection. TNF-neutralizing drugs used to treat inflammatory disorders have been reported to increase the risk of tuberculosis (TB), in accordance with animal studies. The present study takes a computational approach toward characterizing the role of TNF in protection against the tubercle bacillus in both active and latent infection. We extend our previous mathematical models to investigate the roles and production of soluble (sTNF) and transmembrane TNF (tmTNF). We analyze effects of anti-TNF therapy in virtual clinical trials (VCTs) by simulating two of the most commonly used therapies, anti-TNF antibody and TNF receptor fusion, predicting mechanisms that explain observed differences in TB reactivation rates. The major findings from this study are that bioavailability of TNF following anti-TNF therapy is the primary factor for causing reactivation of latent infection and that sTNF—even at very low levels—is essential for control of infection. Using a mathematical model, it is possible to distinguish mechanisms of action of the anti-TNF treatments and gain insights into the role of TNF in TB control and pathology. Our study suggests that a TNF-modulating agent could be developed that could balance the requirement for reduction of inflammation with the necessity to maintain resistance to infection and microbial diseases. Alternatively, the dose and timing of anti-TNF therapy could be modified. Anti-TNF therapy will likely lead to numerous incidents of primary TB if used in areas where exposure is likely.     

The role of cytokines in the pathogenesis of cutaneous lupus erythematosus

Cutaneous lupus erythematosus (CLE) is an inflammatory disease with a broad range of cutaneous manifestations that may be accompanied by systemic symptoms. The pathogenesis of CLE is complex, multifactorial and incompletely defined. Below we review the current understanding of the cytokines involved in these processes. Ultraviolet (UV) light plays a central role in the pathogenesis of CLE, triggering keratinocyte apoptosis, transport of nucleoprotein autoantigens to the keratinocyte cell surface and the release of inflammatory cytokines (including interferons (IFNs), tumor necrosis factor (TNF)-α, interleukin (IL)-1, IL-6, IL-8, IL-10 and IL-17). Increased IFN, particularly type I IFN, is central to the development of CLE lesions. In CLE, type I IFN is produced in response to nuclear antigens, immune complexes and UV light. Type I IFN increases leukocyte recruitment to the skin via inflammatory cytokines, chemokines, and adhesion molecules, thereby inducing a cycle of cutaneous inflammation. Increased TNFα in CLE may also cause inflammation. However, decreasing TNFα with an anti-TNFα agent can induce CLE-like lesions. TNFα regulates B cells, increases the production of inflammatory molecules and inhibits the production of IFN-α. An increase in the inflammatory cytokines IL-1, IL-6, IL-10, IL-17 and IL-18 and a decrease in the anti-inflammatory cytokine IL-12 also act to amplify inflammation in CLE. Specific gene mutations may increase the levels of these inflammatory cytokines in some CLE patients. New drugs targeting various aspects of these cytokine pathways are being developed to treat CLE and systemic lupus erythematosus (SLE).     

Role of tumor necrosis factor in monocyte/macrophage tumor cytotoxicity in vitro

The ability of activated monocytes/macrophages to exert cytotoxic effects in vitro which are preferentially manifest on target cells displaying a transformed phenotype has elicited intense efforts aimed at a molecular characterization of the underlying mechanism. This multistep reaction is typified by an apparently stringent requirement for conjugation between the effector and target to facilitate cytotoxicity, which has therefore long caused bias against the role of soluble effector molecules in mediating target cell damage. However, several laboratories have recently demonstrated a compelling role for at least one such mediator, tumor necrosis factor (TNF), in cell-mediated cytotoxicity exerted against certain target cells; these studies indicated that specific anti-TNF antibodies could block direct monocyte/macrophage-mediated cytotoxicity of TNF-sensitive targets. More recently we have shown that some targets which are completely resistant to soluble or fluid-phase TNF are effectively lysed by a TNF-dependent mechanism upon coculture with activated macrophages under conditions in which conjugation is facilitated. Furthermore, macrophage-mediated cytolysis of both TNF-sensitive and TNF-resistant targets occurs independently of the action of secreted TNF via this mechanism. The purpose of this review is to consider the implications of distinct modes of effector cell delivery of TNF to the target for molecular characterization of the target injury phase of macrophage-mediated tumor cytotoxicity.     

Evaluation of an anti-tumor necrosis factor therapeutic in a mouse model of Niemann-Pick C liver disease

Background

Niemann-Pick type C (NPC) disease is a lysosomal storage disease characterized by the accumulation of cholesterol and glycosphingolipids. The majority of NPC patients die in their teen years due to progressive neurodegeneration; however, half of NPC patients also suffer from cholestasis, prolonged jaundice, and hepatosplenomegaly. We previously showed that a key mediator of NPC liver disease is tumor necrosis factor (TNF) α, which is involved in both proinflammatory and apoptotic signaling cascades. In this study, we tested the hypothesis that blocking TNF action with an anti-TNF monoclonal antibody (CNTO5048) will slow the progression of NPC liver disease.

Methodology/Principal Findings

Treatment of wild-type C57BL/6 mice with NPC1-specific antisense oligonucleotides led to knockdown of NPC1 protein expression in the liver. This caused classical symptoms of NPC liver disease, including hepatic cholesterol accumulation, hepatomegaly, elevated serum liver enzymes, and lipid laden macrophage accumulation. In addition, there was a significant increase in the number of apoptotic cells and a proliferation of stellate cells. Concurrent treatment of NPC1 knockdown mice with anti-TNF had no effect on the primary lipid storage or accumulation of lipid-laden macrophages. However, anti-TNF treatment slightly blunted the increase in hepatic apoptosis and stellate cell activation that was seen with NPC1 knockdown.

Conclusions/Significance

Current therapeutic options for NPC disease are limited. Our results provide proof of principle that pharmacologically blocking the TNF-α inflammatory cascade can slightly reduce certain markers of NPC disease. Small molecule inhibitors of TNF that penetrate tissues and cross the blood-brain barrier may prove even more beneficial.     

TNF and TNFR biology in health and disease.

Many insights have been gained into cytokine-regulated control of inflammatory processes and host defence in recent years. Evidence has also gradually accumulated that cytokine cascades play a central role in events regulating cell death and differentiation. Further developments include an understanding that the biological effects of the tumor necrosis factor-alpha (TNF-alpha or TNFSF) cytokine may be regulated by soluble TNF receptor binding and that modulation of receptor levels may permit physiological inhibition of TNF action. There has been a gradual realisation of the value of TNF/TNFR ratios as predictors of disease outcome, and the discovery of functional regulatory polymorphisms of the TNF gene and mutations of TNFRSF1A (TNFR1 receptor) have led to conceptual breakthroughs in our understanding of the genetic control of inflammation. However the exact mechanisms by which TNFRSF1A mutations give rise to disease susceptibility are not yet well understood. Over the past 10 years these concepts have been used as the basis for successful anti-TNF therapy of autoimmune diseases like rheumatoid arthritis (RA) and Crohn's disease.     

Clinical applications of tumour necrosis factor

Tumour necrosis factor (TNF) is a polypeptide hormone produced in vivo by activated macrophages and lymphocytes. TNF has diverse effects in vivo and has a physiological role as an immune modulator, as a mediator of the immune response, both through activation of neutrophils and eosinophils, and also affects the vascular endothelium. TNF also has antiviral activity and causes alterations in lipid metabolism. In disease states excessive production of TNF may have adverse affects. TNF has been implicated as a mediator of endotoxic shock, inflammatory joint disease, immune deficiency states, allograft rejection, and in the cachexia associated with malignant disease and some parasitic infections. When used in pharmacological doses, TNF is cytotoxic to many malignant cells in vitro and in vivo. The mechanisms underlying cytotoxicity are not fully elucidated but involve both a direct toxic effect to the cell and an indirect effect on tumour vasculature. Cytotoxicity is not universal and TNF may act as a differentiating agent or growth factor for some haematological cell types. So far the clinical application of TNF has been as a treatment for cancer in Phase I and II trials in patients with advanced disease and its efficacy here is still unproven. TNF may have potential for clinical application in combination therapy for cancer. There is experimental evidence for its interaction with other biological agents and cytotoxic drugs. The use of specific antibodies to inhibit production of TNF, or other agents to antagonise the toxic effects of TNF may have clinical relevance in counteracting septic shock and the clinical manifestations of TNF in inflammatory and neoplastic disease.     

Signal transduction by tumor necrosis factor receptors

Tumor necrosis factor (TNF) is a key mediator in the inflammatory response which is implicated in the onset of a number of diseases. Research on TNF led to the characterization of the largest family of cytokines known until now, the TNF superfamily, which exert their biological effects through the interaction with transmembrane receptors of the TNFR superfamily. TNF itself exerts its biological effects interacting with two different receptors: TNFR1 and TNFR2. TNFR1 presents a death domain on its intracellular region. In contrast to TNFR1, TNFR2 does not have a death domain. Activation of TNFR1 implies the consecutive formation of two different TNF receptor signalling complexes. Complex I controls the expression of antiapoptotic proteins that prevent the triggering of cell death processes, whereas Complex II triggers cell death processes. TNFR2 only signals for antiapoptotic reactions. However, recent evidence indicates that TNFR2 also signals to induce TRAF2 degradation. TRAF2 is a key mediator in signal transduction of both TNFR1 and TNFR2. Thus, this novel signalling pathway has two important implications: on one hand, it represents an auto regulatory loop for TNFR2; on the other hand, when this signal is triggered TNFR1 activity is modified so that antiapoptotic pathways are inhibited and apoptotic reactions are enhanced.     

Whole cell-based surface plasmon resonance measurement to assess binding of anti-TNF agents to transmembrane target

We developed a technique for the measurement of surface plasmon resonance (SPR) to detect interactions of anti-tumor necrosis factor (TNF) agents with transmembrane TNF-α (mTNF-α) on living whole cells. The injection of a suspension of mTNF-α expressing Jurkat cells, used as an analyte, gave a clear binding response to anti-TNF agents, such as etanercept, infliximab and adalimumab, immobilized on sensorchip. The binding response of the analyte cells increased in a concentration-dependent manner and was competitively reduced by adding soluble TNF receptors to the analyte cell suspension. Treatment of analyte cells with free anti-TNF agent before injection reduced the binding response between the analyte cells and immobilized-etanercept on sensorchip, and the inhibitory effect of free anti-TNF agent was concordant with the affinity of anti-TNF agent for soluble TNF-α. These findings indicate that the SPR response arises from specific binding between anti-TNF agent and its target on cell membrane.     

Oligodeoxynucleotides enhance lipopolysaccharide-stimulated synthesis of tumor necrosis factor: dependence on phosphorothioate modification and reversal by heparin.

BACKGROUND: Specific inhibition of target proteins by antisense oligodeoxynucleotides is an extensively studied experimental approach. This technique is currently being tested in clinical trials applying phosphorothioate-modified oligonucleotides as therapeutic agents. These polyanionic molecules, however, may also exert non-antisense-mediated effects. MATERIALS AND METHODS: We examined the influence of oligonucleotides on lipopolysaccharide (LPS)-stimulated tumor necrosis factor alpha (TNF alpha) synthesis in freshly isolated human peripheral blood mononuclear cells. Oligonucleotides (18 mer) with different degrees of phosphorothioate modification were studied. RESULTS: The addition of phosphorothioate oligonucleotides (5 microM) caused amplification of TNF synthesis of up to 410% compared with the control with LPS alone. Without LPS stimulation, phosphorothioate oligonucleotides did not induce TNF production. We demonstrate that the enhancement of LPS-stimulated TNF production by phosphorothioate oligonucleotides does not rely on the intracellular presence of oligonucleotides and is not mediated by LPS contamination. Partially phosphorothioate-modified oligonucleotides and unmodified oligonucleotides did not increase TNF synthesis. High concentrations of the polyanion heparin reversed the oligonucleotide-induced enhancement of TNF synthesis. CONCLUSIONS: The data suggest that amplification of TNF synthesis may be caused by binding of the polyanionic phosphorothioate oligonucleotide to cationic sites on the cell surface. Such binding sites have been proposed for polyanionic glycoaminoglycans of the extracellular matrix, which have also been described to augment LPS-stimulated TNF synthesis. The present results are relevant to all in vitro studies attempting to influence protein synthesis in monocytes by using phosphorothioate oligonucleotides. The significance of our findings for in vivo applications of phosphorothioates in situations where there is a stimulus for TNF synthesis, such as in sepsis, should be elucidated.     

Thalidomide as an anti-cancer agent

Thalidomide is a glutamic acid derivative initially introduced as a sedative hypnotic nearly forty years ago. It was withdrawn following numerous reports linking it to a characteristic pattern of congenital abnormalities in babies born to mothers who used the drug for morning sickness. It has gradually been re-introduced into clinical practice over the past two decades, albeit under strict regulation, since it was found to be useful in the management of erythema nodosum leprosum and HIV wasting syndrome. Recognition of its anti-angiogenic effect led to its evaluation in the treatment of various malignancies, where angiogenesis has been shown to play an important role. Numerous clinical trials done over the past four years have confirmed the significant anti-myeloma activity of this drug. It has also shown promise in preliminary trials in the treatment of a variety of different malignant diseases. The mechanisms of its antineoplastic effects continue to be the focus of ongoing research. It has become clear that even though its anti angiogenic effects play a significant role in the anti-tumor activity, there are other properties of this drug which are responsible as well. It also possesses anti-TNF alpha activity, which has led to its evaluation in several inflammatory states. In this concise review, we briefly describe the historical background and pharmacological aspects of this drug. We have concisely reviewed the current knowledge regarding mechanisms of its anti-neoplastic activity and the results of various clinical trials in oncology.     

Biological functions of tumor necrosis factor cytokines and their receptors

Tumor necrosis factor (TNF; formerly known as TNFalpha) and lymphotoxin (LT)alpha, originally characterized by their ability to induce tumor cell apoptosis and cachexia, are now considered as central mediators of a broad range of biological activities. These activities encompass beneficial effects for the host in inflammation and in protective immune responses against a variety of infectious pathogens. TNF family members on the other hand also exert host-damaging effects in sepsis, in tumor cachexia as well as in autoimmune diseases. In addition, the essential roles of the core members of the TNF superfamily, LTalpha, LTbeta, TNF, and LIGHT as well as their receptors during the organogenesis of secondary lymphoid organs and the maintenance of the architecture of lymphatic tissues now becomes appreciated. The elucidation of the biological functions of these cytokines and their specific cell surface receptors has been crucially advanced by the study of gene-targeted mouse strains. This presentation summarizes the roles of TNFR and TNF-like cytokines in infection, sepsis and autoimmunity as well as the pivotal involvement of these molecules in the development of secondary lymphoid organs.     

IL 3 and IL 6 do not induce bone resorption in vitro

Bone resorption in vitro and in vivo can be induced by interleukin 1 (IL 1) and tumor necrosis factor (TNF), both of which are potent inflammatory cytokines. Additionally, there are other factors produced by cells which can active osteoclasts. Because diverse factors are involved in bone resorption, we examined the role of two other inflammatory cytokines, IL 3 and IL 6. IL 3 has been shown to induce the formation of osteoclast-like cells from precursors, while IL 6 is a potent mediator of inflammatory responses. Osteoclast activity in neonatal mouse calvaria was measured as 45Ca released into the supernatant fluid following a 48 hr incubation period with cytokine. Our results show that while parathyroid hormone (PTH) and IL 1 are potent inducers of bone resorption, neither IL 3 nor IL 6 displayed such activity.