Blake Pepinsky

LINGO-1 antagonist promotes spinal cord remyelination and axonal integrity in MOG-induced experimental autoimmune encephalomyelitis

Demyelinating diseases, such as multiple sclerosis, are characterized by the loss of the myelin sheath around neurons, owing to inflammation and gliosis in the central nervous system (CNS). Current treatments therefore target anti-inflammatory mechanisms to impede or slow disease progression. The identification of a means to enhance axon myelination would present new therapeutic approaches to inhibit and possibly reverse disease progression. Previously, LRR and Ig domain–containing, Nogo receptor–interacting protein (LINGO-1) has been identified as an in vitro and in vivo negative regulator of oligodendrocyte differentiation and myelination. Here we show that loss of LINGO-1 function by Lingo1 gene knockout or by treatment with an antibody antagonist of LINGO-1 function leads to functional recovery from experimental autoimmune encephalomyelitis. This is reflected biologically by improved axonal integrity, as confirmed by magnetic resonance diffusion tensor imaging, and by newly formed myelin sheaths, as determined by electron microscopy. Antagonism of LINGO-1 or its pathway is therefore a promising approach for the treatment of demyelinating diseases of the CNS.

Blockade of Nogo-66, Myelin-Associated Glycoprotein, and Oligodendrocyte Myelin Glycoprotein by Soluble Nogo-66 Receptor Promotes Axonal Sprouting and Recovery after Spinal Injury

The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind to the Nogo-66 receptor (NgR) and inhibit axonal growth in vitro. Transgenic or viral blockade of NgR function allows axonal sprouting in vivo. Here, we administered the soluble function-blocking NgR ectodomain [aa 27-310; NgR(310)ecto] to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.

Promotion of central nervous system remyelination by induced differentiation of oligodendrocyte precursor cells

Repair of demyelinated axons in diseases such as multiple sclerosis requires activation of the myelination program in existing or newly recruited oligodendrocyte precursor cells (OPCs). The control of OPC differentiation and initiation of myelination during repair is poorly understood. In this study, we test the ability of anti–LINGO‐1 reagents to promote myelination in vitro and remyelination in the rodent adult central nervous system in vivo.

Multiple actions of systemic artemin in experimental neuropathy

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury–induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

A neutralizing anti-Nogo66 receptor monoclonal antibody reverses inhibition of neurite outgrowth by central nervous system myelin.

The Nogo66 receptor (NgR1) is a neuronal, leucinerich repeat (LRR) protein that binds three central nervous system (CNS) myelin proteins, Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein, and mediates their inhibitory effects on neurite growth. Although the LRR domains on NgR1 are necessary for binding to the myelin proteins, the exact epitope(s) involved in ligand binding is unclear. Here we report the generation and detailed characterization of an anti-NgR1 monoclonal antibody, 7E11. The 7E11 monoclonal antibody blocks Nogo, myelin-associated glycoprotein, and oligodendrocyte myelin glycoprotein binding to NgR1 with IC50 values of 120, 14, and 4.5 nm, respectively, and effectively promotes neurite outgrowth of P3 rat dorsal root ganglia neurons cultured on a CNS myelin substrate. Further, we have defined the molecular epitope of 7E11 to be DNAQLR located in the third LRR domain of rat NgR1. Our data demonstrate that anti-NgR1 antibodies recognizing this epitope, such as 7E11, can neutralize CNS myelin-dependent inhibition of neurite outgrowth. Thus, specific anti-NgR1 antibodies may represent a useful therapeutic approach for promoting CNS repair after injury.

A novel platform for engineering blood-brain barrier-crossing bispecific biologics

The blood‐brain barrier (BBB) prevents the access of therapeutic antibodies to central nervous system (CNS) targets. The engineering of bispecific antibodies in which a therapeutic “arm” is combined with a BBB‐transcytosing arm can significantly enhance their brain delivery. The BBB‐permeable single‐domain antibody FC5 was previously isolated by phenotypic panning of a naive llama single‐domain antibody phage display library. In this study, FC5 was engineered as a mono‐ and bivalent fusion with the human Fc domain to optimize it as a modular brain delivery platform. In vitro studies demonstrated that the bivalent fusion of FC5 with Fc increased the rate of transcytosis (Papp) across brain endothelial monolayer by 25% compared with monovalent fusion. Up to a 30‐fold enhanced apparent brain exposure (derived from serum and cerebrospinal fluid pharmacokinetic profiles) of FC5‐compared with control domain antibody‐Fc fusions after systemic dosing in rats was observed. Systemic pharmacological potency was evaluated in the Hargreaves model of inflammatory pain using the BBB‐impermeable neuropeptides dalargin and neuropeptide Y chemically conjugated with FC5‐Fc fusion proteins. Improved serum pharmacokinetics of Fc‐fused FC5 contributed to a 60‐fold increase in pharmacological potency compared with the single‐domain version of FC5; bivalent and monovalent FC5 fusions with Fc exhibited similar systemic pharmacological potency. The study demonstrates that modular incorporation of FC5 as the BBB‐carrier arm in bispecific antibodies or antibody‐drug conjugates offers an avenue to develop pharmacologically active biotherapeutics for CNS indications.—Farrington, G. K., Caram‐Salas, N., Haqqani, A. S., Brunette, E., Eldredge, J., Pepinsky, B., Antognetti, G., Baumann, E., Ding, W., Garber, E., Jiang, S., Delaney, C., Boileau, E., Sisk, W. P., Stanimirovic, D. B., A novel platform for engineering blood‐brain barrier‐crossing bispecific biologics. FASEB J. 28, 4764–4778 (2014). www.fasebj.org

Tat-Mediated Protein Delivery Can Facilitate MHC Class I Presentation of Antigens

We have previously shown that the tat protein of HIV-1 can be used as a carrier to promote the intracellular delivery of heterologous proteins. Here we have tested if the tat-delivery technology can be used to direct MHC class I presentation of native protein, using ovalbumin (OVA) as a model system. We show that a tat-ovalbumin conjugate (tatOVA) can be delivered into cells and that subsequent processing and presentation occurs, resulting in effective and specific killing of these target cells by an OVA specific cytotoxic T-lymphocyte (CTL) line. Comparison with the E.G7 line that expresses the OVA gene indicates that tat-mediated delivery is as efficient as endogenous expression in this system. Tat-mediated antigenic protein delivery may be useful both as a research technique and, potentially, as a therapeutic or prophylactic vaccine.

Inhibition of LINGO-1 promotes functional recovery after experimental spinal cord demyelination.

Blocking LINGO-1 has been shown to enhance remyelination in the rat lysolecithin-induced focal spinal cord demyelination model. We used transcranial magnetic motor-evoked potentials (tcMMEPs) to assess the effect of blocking LINGO-1 on recovery of axonal function in a mouse lysolecithin model at 1, 2 and 4weeks after injury. The role of LINGO-1 was assessed using LINGO-1 knockout (KO) mice and in wild-type mice after intraperitoneal administration of anti-LINGO-1 antagonist monoclonal antibody (mAb3B5). Response rates (at 2 and 4weeks) and amplitudes (at 4weeks) were significantly increased in LINGO-1 KO and mAb3B5-treated mice compared with matched controls. The latency of potentials at 4weeks was significantly shorter in mAb3B5-treated mice compared with controls. Lesion areas in LINGO-1 KO and mAb3B5-treated mice were reduced significantly compared with matched controls. The number of remyelinated axons within the lesions was increased and the G-ratios of the axons were decreased in both LINGO-1 KO and mAb3B5-treated mice compared with matched controls. These data provide morphometric and functional evidence of enhancement of remyelination associated with antagonism of LINGO-1.

LINGO-1 regulates oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts

Oligodendrocyte differentiation is negatively regulated by LINGO-1 and positively regulated by the ErbB2 receptor tyrosine kinase. In wild-type oligodendrocytes, inhibition of ErbB2 blocks differentiation, whereas activation of ErbB2 promotes differentiation. In LINGO-1(-/-) oligodendrocytes, inhibition of ErbB2 blocks oligodendrocyte differentiation; whereas activation of ErbB2 does not enhance differentiation. Biological and biochemical evidence showing that LINGO-1 can directly bind to ErbB2, block ErbB2 translocation into lipid rafts, and inhibit its phosphorylation for activation. The study demonstrates a novel regulatory mechanism of ErbB2 function whereby LINGO-1 suppresses oligodendrocyte differentiation by inhibiting ErbB2 translocation and activation in lipid rafts.

Oligodendrocyte differentiation and myelination defects in OMgp null mice

OMgp is selectively expressed in CNS by oligodendrocyte. However, its potential role(s) in oligodendrocyte development and myelination remain unclear. We show that OMgp null mice are hypomyelinated in their spinal cords, resulting in slower ascending and descending conduction velocities compared to wild-type mice. Consistent with the hypomyelination, in the MOG induced EAE model, OMgp null mice show a more severe EAE clinical disease and slower nerve conduction velocity compared to WT animals. The contribution of OMgp to oligodendrocyte differentiation and myelination was verified using cultured oligodendrocytes from null mice. Oligodendrocytes isolated from OMgp null mice show a significant decrease in the number of MBP(+) cells and in myelination compared to wild-type mice. The dramatic effects of the OMgp KO in oligodendrocyte maturation in vivo and in vitro reveal a new and important function for OMgp in regulating CNS myelination.