RESUMO
LINGO-1 (leucine-rich repeat and Ig domain containing NOGO receptor interacting protein-1) is a negative regulator of myelination and repair of damaged axons in the central nervous system (CNS). Blocking LINGO-1 function leads to robust remyelination. The anti-LINGO-1 Li81 antibody is currently being evaluated in clinical trials for multiple sclerosis (MS) and is the first MS therapy that directly targets myelin repair. LINGO-1 is selectively expressed in brain and spinal cord but not in peripheral tissues. Perhaps the greatest concern for Li81 therapy is the limited access of the drug to the CNS. Here, we measured Li81 concentrations in brain, spinal cord, and cerebral spinal fluid in rats after systemic administration and correlated them with dose-efficacy responses in rat lysolecithin and experimental autoimmune encephalomyelitis spinal cord models of remyelination. Remyelination was dose-dependent, and levels of Li81 in spinal cord that promoted myelination correlated well with affinity measurements for the binding of Li81 to LINGO-1. Observed Li81 concentrations in the CNS of 0.1 to 0.4% of blood levels are consistent with values reported for other antibodies. To understand the features of the antibody that affect CNS penetration, we also evaluated the pharmacokinetics of Li81 Fab2, Fab, and poly(ethylene glycol)-modified Fab. The reagents all showed similar CNS exposure despite large differences in their sizes, serum half-lives, and volumes of distribution, and area under the curve (AUC) measurements in the CNS directly correlated with AUC measurements in serum. These studies demonstrate that exposure levels achieved by passive diffusion of the Li81 monoclonal antibody into the CNS are sufficient and lead to robust remyelination.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/farmacologia , Encefalomielite Autoimune Experimental/tratamento farmacológico , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Proteínas do Tecido Nervoso/imunologia , Medula Espinal/efeitos dos fármacos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Líquido Cefalorraquidiano/química , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Encefalomielite Autoimune Experimental/metabolismo , Feminino , Lisofosfatidilcolinas , Masculino , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Regeneração , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
The use of LINGO-1 antagonists to promote repair of damaged myelin is an emerging therapeutic opportunity for treatment of CNS diseases caused by demyelination such as multiple sclerosis. The Li33 anti-LINGO-1 antibody is a potent inducer of myelination in vitro and in vivo, but aggregation issues prevented the engineering of an optimal development candidate. PEGylated Li33 Fab' is one of several versions of the Li33 antibody that is being investigated in an attempt to identify the most favorable anti-LINGO-1 antibody design. For targeted PEGylation, a Li33 Fab' construct was engineered with a single unpaired cysteine in the heavy-chain hinge sequence. The Fab' was expressed in CHO cells, purified, and PEGylated with 20 kDa methoxy-poly(ethylene glycol) maleimide using a reaction strategy optimized to improve the yield of the PEG-Fab'. Biochemical analysis of the Li33 PEG-Fab' verified the selectivity of the PEGylation reaction. The in vitro and in vivo attributes of the PEG-Fab' were benchmarked against a Li33 full antibody. Both the Li33 PEG-Fab' and intact antibody bound LINGO-1 with nanomolar affinity, promoted myelination in an in vitro signaling assay, and promoted the repair of damaged myelin in the rat lysolecithin model. These studies extend our understanding of the biological activity of the Li33 mAb and validate the use of an anti-LINGO-1 PEG-Fab' for treatment of CNS diseases caused by demyelination.
Assuntos
Anticorpos Monoclonais/química , Fragmentos Fab das Imunoglobulinas/química , Polietilenoglicóis/química , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Cricetinae , Cricetulus , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/isolamento & purificação , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Modelos Animais , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , RatosRESUMO
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.
Assuntos
Proteínas do Tecido Nervoso/farmacologia , Dor/tratamento farmacológico , Nervos Espinhais/lesões , Animais , Biomarcadores , Peptídeo Relacionado com Gene de Calcitonina/efeitos dos fármacos , Dinorfinas/efeitos dos fármacos , Masculino , Ratos , Nervos Espinhais/efeitos dos fármacosRESUMO
NgRI (Nogo-66 receptor) is part of a signalling complex that inhibits axon regeneration in the central nervous system. Truncated soluble versions of NgRI have been used successfully to promote axon regeneration in animal models of spinal-cord injury, raising interest in this protein as a potential therapeutic target. The LRR (leucine-rich repeat) regions in NgRI are flanked by N- and C-terminal disulfide-containing 'cap' domains (LRRNT and LRRCT respectively). In the present work we show that, although functionally active, the NgRI(310)-Fc fusion protein contains mislinked and heterogeneous disulfide patterns in the LRRCT domain, and we report the generation of a series of variant molecules specifically designed to prevent this heterogeneity. Using these variants we explored the effects of modifying the NgRI truncation site or the spacing between the NgRI and Fc domains, or replacing cysteines within the NgRI or IgG hinge regions. One variant, which incorporates replacements of Cys²66 and Cys³°9 with alanine residues, completely eliminated disulfide scrambling while maintaining functional in vitro and in vivo efficacy. This modified NgRI-Fc molecule represents a significantly improved candidate for further pharmaceutical development, and may serve as a useful model for the optimization of other IgG fusion proteins made from LRR proteins.
Assuntos
Dissulfetos/metabolismo , Proteínas da Mielina/química , Engenharia de Proteínas/métodos , Receptores de Superfície Celular/química , Proteínas Recombinantes de Fusão/química , Sequência de Aminoácidos , Animais , Comportamento Animal/efeitos dos fármacos , Cristalografia por Raios X , Modelos Animais de Doenças , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Humanos , Masculino , Anotação de Sequência Molecular , Dados de Sequência Molecular , Proteínas da Mielina/genética , Receptor Nogo 1 , Estabilidade Proteica , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Traumatismos da Medula Espinal/tratamento farmacológico , Raízes Nervosas Espinhais/lesõesRESUMO
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.
Assuntos
Axônios/fisiologia , Proteínas da Mielina/antagonistas & inibidores , Glicoproteína Associada a Mielina/antagonistas & inibidores , Glicoproteína Associada a Mielina/metabolismo , Receptores de Peptídeos/fisiologia , Traumatismos da Medula Espinal/patologia , Animais , Axônios/metabolismo , Potencial Evocado Motor , Feminino , Proteínas Ligadas por GPI , Injeções Espinhais , Atividade Motora , Glicoproteína Mielina-Oligodendrócito , Proteínas Nogo , Receptor Nogo 1 , Oligodendroglia/metabolismo , Fragmentos de Peptídeos/administração & dosagem , Fragmentos de Peptídeos/farmacologia , Ratos , Ratos Sprague-Dawley , Receptores de Superfície Celular , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/farmacologia , Serotonina/metabolismo , Solubilidade , Medula Espinal/fisiopatologia , Medula Espinal/ultraestrutura , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
Survival and differentiation of oligodendrocytes are important for the myelination of central nervous system (CNS) axons during development and crucial for myelin repair in CNS demyelinating diseases such as multiple sclerosis. Here we show that death receptor 6 (DR6) is a negative regulator of oligodendrocyte maturation. DR6 is expressed strongly in immature oligodendrocytes and weakly in mature myelin basic protein (MBP)-positive oligodendrocytes. Overexpression of DR6 in oligodendrocytes leads to caspase 3 (casp3) activation and cell death. Attenuation of DR6 function leads to enhanced oligodendrocyte maturation, myelination and downregulation of casp3. Treatment with a DR6 antagonist antibody promotes remyelination in both lysolecithin-induced demyelination and experimental autoimmune encephalomyelitis (EAE) models. Consistent with the DR6 antagoinst antibody studies, DR6-null mice show enhanced remyelination in both demyelination models. These studies reveal a pivotal role for DR6 signaling in immature oligodendrocyte maturation and myelination that may provide new therapeutic avenues for the treatment of demyelination disorders such as multiple sclerosis.
Assuntos
Bainha de Mielina/metabolismo , Oligodendroglia/metabolismo , Receptores do Fator de Necrose Tumoral/fisiologia , Animais , Western Blotting , Caspase 3/metabolismo , Caspase 3/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Sobrevivência Celular/fisiologia , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/fisiopatologia , Encefalomielite Autoimune Experimental/terapia , Ativação Enzimática , Regulação da Expressão Gênica , Camundongos , Esclerose Múltipla/metabolismo , Esclerose Múltipla/terapia , Bainha de Mielina/fisiologia , Oligodendroglia/fisiologia , Ratos , Receptores do Fator de Necrose Tumoral/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Monoclonal antibodies (Mabs) are a favorite drug platform of the biopharmaceutical industry. Currently, over 20 Mabs have been approved and several hundred others are in clinical trials. The anti-LINGO-1 Mab Li33 was selected from a large panel of antibodies by Fab phage display technology based on its extraordinary biological activity in promoting oligodendrocyte differentiation and myelination in vitro and in animal models of remyelination. However, the Li33 Fab had poor solubility when converted into a full antibody in an immunoglobulin G1 framework. A detailed analysis of the biochemical and structural features of the antibody revealed several possible reasons for its propensity to aggregate. Here, we successfully applied three molecular approaches (isotype switching, targeted mutagenesis of complementarity determining region residues, and glycosylation site insertion mutagenesis) to address the solubility problem. Through these efforts we were able to improve the solubility of the Li33 Mab from 0.3 mg/mL to >50 mg/mL and reduce aggregation to an acceptable level. These strategies can be readily applied to other proteins with solubility issues.
Assuntos
Anticorpos Monoclonais/química , Imunoglobulina G/química , Proteínas de Membrana/imunologia , Mutagênese Sítio-Dirigida/métodos , Proteínas do Tecido Nervoso/imunologia , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Área Sob a Curva , Cristalografia por Raios X , Eletroforese em Gel de Poliacrilamida , Switching de Imunoglobulina , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/genética , Fragmentos Fab das Imunoglobulinas/metabolismo , Imunoglobulina G/genética , Imunoglobulina G/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Estabilidade Proteica , Solubilidade , TemperaturaRESUMO
Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.
Assuntos
Dissulfetos/química , Leucina/química , Sequências Repetitivas de Aminoácidos , Alquilação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Proteínas Ligadas por GPI , Humanos , Espectrometria de Massas , Modelos Moleculares , Dados de Sequência Molecular , Proteínas da Mielina , Receptor Nogo 1 , Mapeamento de Peptídeos , Conformação Proteica , Receptores de Superfície Celular , Proteínas Recombinantes/química , Tripsina/químicaRESUMO
Methylprednisolone (MP) is a synthetic glucocorticoid used for the treatment of spinal cord injury (SCI). Soluble Nogo-66 receptor (NgR) ectodomain is a novel experimental therapy for SCI that promotes axonal regeneration by blocking the growth inhibitory effects of myelin constituents in the adult central nervous system. To evaluate the potential complementarity of these mechanistically distinct pharmacological reagents we compared their effects alone and in combination after thoracic (T7) dorsal hemisection in the rat. Treatment with an ecto-domain of the rat NgR (27-310) fused to a rat IgG [NgR(310)ecto-Fc] (50 microm intrathecal, 0.25 microL/h for 28 days) or MP alone (30 mg/kg i.v., 0, 4 and 8 h postinjury) improved the rate and extent of functional recovery measured using Basso, Beattie, Bresnahan (BBB) scoring and footprint analysis. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NgR(310)ecto-Fc was not apparent until 2 weeks after SCI. NgR(310)ecto-Fc or MP treatment resulted in increased axonal sprouting and/or regeneration, quantified by counting biotin dextran amine-labeled corticospinal tract axons, and increased the number of axons contacting motor neurons in the ventral horn gray matter caudal to the lesion. Combined treatment with NgR(310)ecto-Fc and MP had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone. The data demonstrate that NgR(310)ecto-Fc and MP act in a temporally and mechanistically distinct manner and suggest that they may have complementary effects.
Assuntos
Metilprednisolona/uso terapêutico , Receptores de Peptídeos/uso terapêutico , Traumatismos da Medula Espinal/tratamento farmacológico , Análise de Variância , Animais , Axônios/efeitos dos fármacos , Axônios/fisiologia , Comportamento Animal , Biotina/análogos & derivados , Biotina/metabolismo , Células Cultivadas , Embrião de Galinha , Dextranos/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Quimioterapia Combinada , Comportamento Exploratório/efeitos dos fármacos , Feminino , Proteínas Ligadas por GPI , Gânglios Espinais/citologia , Imunoglobulina G/uso terapêutico , Laminectomia/métodos , Proteínas da Mielina , Bainha de Mielina/metabolismo , Regeneração Nervosa/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Receptor Nogo 1 , Tratos Piramidais/efeitos dos fármacos , Tratos Piramidais/metabolismo , Ratos , Ratos Long-Evans , Receptores de Superfície Celular , Receptores de Peptídeos/biossíntese , Receptores de Peptídeos/química , Receptores de Peptídeos/imunologia , Proteínas Recombinantes/uso terapêutico , Recuperação de Função Fisiológica/efeitos dos fármacos , Traumatismos da Medula Espinal/fisiopatologiaRESUMO
To clarify whether glial cell line-derived neurotrophic factor (GDNF) receptor alpha-1 (GFRalpha1), the glycosylphosphatidylinositol (GPI)-linked coreceptor for GDNF, is also a functional coreceptor for artemin (ART), we have studied receptor binding, signaling, and neuronal survival. In cell-free binding studies, GFRalpha1-Ig displayed strong preferential binding to GDNF, though in the presence of soluble RET, weak binding to ART could also be detected. However, using GFRalpha1-transfected NB41A3 cells, ART showed no detectable competition against the binding of (125)I-labeled GDNF. Moreover, ART failed to induce phosphorylation of extracellular signal-related kinase (ERK) and Akt in these cells and was >10(4)-fold less potent than GDNF in stimulating RET phosphorylation. When rat primary dorsal root ganglion (DRG) neurons were used, only the survival promoting activity of GDNF and not that of ART was blocked by an anti-GFRalpha1 antibody. These results indicate that although ART can interact weakly with soluble GFRalpha1 constructs under certain circumstances in vitro, in cell-based functional assays GFRalpha1 is at least 10 000-fold selective for GDNF over ART. The extremely high selectivity of GFRalpha1 for GDNF over ART and the low reactivity of ART for this receptor suggest that GFRalpha1 is not likely to be a functional coreceptor for ART in vivo.
Assuntos
Fatores de Crescimento Neural/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Sobrevivência Celular/fisiologia , Gânglios Espinais/citologia , Gânglios Espinais/fisiologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Camundongos , Fatores de Crescimento Neural/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neurônios Aferentes/citologia , Neurônios Aferentes/fisiologia , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ret , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/fisiologia , Transdução de Sinais/genética , Soluções , Ressonância de Plasmônio de Superfície , TransfecçãoRESUMO
The Nogo66 receptor (NgR1) is a neuronal, leucine-rich 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.