Axonal regeneration after injury within the mammalian central nervous system (CNS) is almost always impossible; the outcome depends on the balance between the intrinsic ability of the nerve fibers in the CNS to re-grow, and the inhibitory factors within the CNS, localized in the microenvironment of the lesion site, which actively prevent the re-growth, and thus the regeneration of the injured fiber tracts.
It has been established that CNS myelin, generated by oligodendrocytes, is the most relevant non-permissive factor for axonal growth in the early phase of an injury, by causing growth cone collapse in vitro as well as in vivo, which results in the direct inhibition of axon outgrowth (for review see: Lee et al., 2003). Only recently major inhibitory factors on CNS myelin have been identified: oligodendrocyte myelin glycoprotein (OMgp), Myelin associated glycoprotein (MAG) and Nogo-A (Domeniconi et al., 2002; reviews: Woolf & Bloechinger, 2002; McGee & Strittmatter, 2003; Lee et al., 2003). The latter protein contains a domain Nogo-66; GrandPré et al., 2000), which exerts a main inhibitory function. Interestingly, all three inhibitory proteins show high expression levels in the CNS and interact with the same neuronal glycosylphosphatidylinositol (GPI) moiety-anchored receptor, the Nogo-66 receptor, or NgR (Fournier et al., 2001). The Nogo-66 receptor, NgR, is a 473 amino acid glycosylphosphatidylinositol-linked protein. It consists of an N-terminal signal sequence, followed by 8 leucine-rich repeat domains, a leucine-rich repeat C-terminal domain (together forming the so-called ectodomain) and the GPI-anchoring domain. Through the GPI-anchor, NgR is linked to the external neuronal plasmalemma.
NgR itself belongs to a family of three CNS-enriched GPI-anchored proteins (named NgR, NgR2 and NgR3) with about 400 sequence identity but very similar overall structural organization (Barton et al. 2003; Lauren et al. 2003; Pignot et al. 2003). Although NgR is the only member known to interact with multiple myelin-associated inhibitory molecules, MAG has recently been shown also to interact with NgR2 (Venkatesh et al. 2005). The function of the NgR homologues is currently not known. NgR itself is not expressed during early development in rodents or chick, but shows high expression levels in adult animals; NgR is expressed in most if not all of the CNS regions, including the spinal cord (Hunt et al., 2002a,b). Spinal cord expression has been shown in chick (Fournier et al., 2001), rat (Hunt et al., 2002a) and mouse (Wang et al., 2002b) at both the mRNA and protein level. Within adult CNS tissue, NgR protein is expressed in all mature neurons, including their axonal processes. Ligand binding to NgR initiates an intracellular signaling cascade, which results in axon outgrowth inhibition and growth cone collapse. As NgR does not contain a transmembrane domain, signaling requires a co-receptor, which transduces the NgR/ligand interaction signal into the cell. The initial step in NgR signaling is its interaction with the co-receptors p75 or TROY (Wong et al., 2002; Shao et al., 2005; Park et al., 2005). A second co-receptor has been identified, called Lingo-1. Only a ternary complex between NgR, P75 or TROY and Lingo-1 constitutes the functional signaling complex (Mi et al., 2004; Park et al., 2005). The outcome of this signaling is a rearrangement of the actin cytoskeleton. In the neuron this actin cytoskeletal change causes an inhibition of axon outgrowth and induction of growth cone collapse.
In vitro, dorsal root ganglion cells from NgR (−/−) mice loose Nogo66 binding capacity and are less responsive to the inhibitory effects of Nogo66, Fc-MAG, OMgp or myelin in a growth cone collapse assay (Kim et al., 2004). NgR (−/−) mice demonstrated increased regeneration of brainstem tracts, including rubrospinal and raphespinal tracts, after partial or complete spinal cord injury. Even after a complete experimental transection of the spinal cord, the NgR (−/−) mice showed increased functional recovery in an open field test. Following hemisection and complete transection of the spinal cord, recovery in NgR (−/−) mice was significantly better than in homozygous (+/+) and heterozygous littermates (Kim et al., 2004).
The present application describes the generation of neutralizing monoclonal antibodies against the NgR, which selectively compete for Nogo-66 binding and that are expected to ameliorate disorders in which NgR activity may be detrimental. The neutralizing monoclonal antibodies of the present invention are expected, for example, to promote neuronal regeneration in the injured CNS, specifically after acute spinal cord injury, brain trauma or neurodegenerative diseases such as for example, Huntington's chorea, Parkinson's disease, Alzheimer's disease or multiple sclerosis.
USPTO Backgrounds
USPTO Backgrounds