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In the context of the above project consortium, the several project groups of IZI (Neurobiology, TNF Signalling, Antibody Engineering, Nanocytes, Designer Cytokines) will actively participate in topic 3 and focus on the role of TNF in MS and the development of novel therapeutic approaches targeting TNFRs.
TNF, a master proinflammatory cytokine, plays a dominant role in the initiation and perpetuation of chronic inflammation, such as that associated with immune-mediated diseases of the CNS. Surprisingly however, clinical trials with TNF neutralizing reagents in MS patients failed to ameliorate disease and in some cases even led to disease exacerbation. Significant beneficial effects of TNF in the CNS have subsequently been revealed. While TNF remains a primary therapeutic target for the treatment of neuroimmune diseases, its targeting should be strictly selective. Specifically, in our previous work TNFR2 signalling has been shown to convey neuroprotection in models of acute neurodegeneration. The TNFR1, on the other hand, has been described to have both neuroprotective and neurotoxic effects. This apparent ambiguity of TNFR1 function can be reconciled in at least two ways. First, unlike TNFR2, TNFR1 cannot protect neurons from glutamate-mediated excitotoxicity (Marchetti et al., J. Biol. Chem. 2004, 279: 32869), which is a dominant mechanism contributing to acute neuron death under conditions of brain ischemia and traumatic brain injury. Second, TNFR1 can indirectly exacerbate axonal and neuronal damage through its potent pro-inflammatory effects, and these become particularly obvious under chronic inflammatory situations (work by Akassoglou et al., 1998, Am. J. Pathol. 153: 801). Based on the above findings, agents that will selectively target one or the other TNF receptor to block inflammation, and selectively promote neuroprotective function, have significant therapeutic potential for neuroinflammatory diseases and are being developed.
In particular, we aim to achieve neuroprotection by selective stimulation of TNFR2. Genetic engineering combined with nanocarrier technology forms the basis to design new classes of TNF receptor selective reagents. We have constructed a novel "single chain" TNF molecule (scTNF) with superior properties with respect to stability and covalent coupling to matrices. These constructs form the basis for the fusion proteins containing an amino-terminal immobilization domain recognizing neuronal cells and, as carboxy-terminal domain, a TNFR2 selective TNF or scTNF mutant. TNFR2 selectivity is ensured by introducing mutations in the TNF molecule; local activation is reached by the specificity of the immobilization domain and the fact that only the cell surface immobilized TNF moiety will be bioactive. This principle will be applied for creation of TNFR2 specific TNF targeted to neuronal tissues by use of appropriate neuro-specific recombinant antibodies including those targeting oligodendrocytes.
A second strategy makes use of a covalent, oriented coupling of TNF to nanoparticle carriers, thereby mimicking a membrane expressed TNF molecule. Using this technology for a TNFR2 specific mutein, a specifically TNFR2 activating nanoparticle will be created. The conceptional advantage of this approach is several fold: Nanoparticles can be sequentially conjugated at the surface with additional reactants, e.g. to enhance BBB passage and/or targeting damaged brain tissue; they can be used in parallel for encapsulation of other therapeutics; they differ in pharmacokinetic properties from soluble protein/peptide therapeutics and show a size dependent capability to enter perivascular space and intracellular uptake via an endocytosis/lysosomal dependent or independent pathway, offering alternative routes of drug delivery and/or signal propagation.
As a complementary approach to modulate TNF signals, blockade of TNFR1 via receptor antagonists as an anti-inflammatory strategy will be pursued. We have previously generated a human TNFR1 specific antagonistic reagent as a potential therapeutic. In addition, murine TNFR specific antibodies suited for established murine EAE models will be also available, allowing an immediate exploitation of the in vivo efficacy of these reagents.
The expected function of the above mentioned designer molecules will be tested in a hierachical order with increasing complexity of the models first in in vitro systems of established cell lines and then primary neuronal and oligodendrocyte cultures exposed to a toxic insult. Successful candidate molecules can be produced in a preclinical scale and analyzed in EAE and ischemia disease models in collaboration with partner laboratories within the consortium. To investigate the functionality of human specific protein therapeutics in vivo, humanized TNFR1 and TNFR2 mouse lines will also be generated by knock-in strategies by these partners.
NeuroproMiSe project presentation: [here]
NeuroproMiSe web-site: http://www.neuropromise.org
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Despite intensive research on the causes and pathogenesis of multiple sclerosis (MS), the genetic basis of this common cause of neurological disability is still unknown and the complex immunopathological mechanisms underlying brain injury are only beginning to be understood. To date, no curative therapy is available and none of the existing drugs can stop the neurodegenerative process responsible for disease progression.The NeuroproMiSe project represents a highly focussed and integrated effort to investigate in depth the genetic and mechanistic pathways involved in inflammation-induced neurodegeneration, and exploit this knowledge to develop novel candidate drugs for effective neuroprotective therapy. NeuroproMiSe specific objectives are: 
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