Nonclinical safety evaluation, pharmacokinetics, and target engagement of Lu AF82422, a monoclonal IgG1 antibody against alpha-synuclein in development for treatment of synucleinopathies.

Alpha-synuclein is a 15 kDa protein associated with neurodegenerative diseases such as Parkinson disease and multiple-system atrophy where pathological forms of alpha-synuclein aggregate and become neurotoxic. Here we describe the nonclinical program to support a first-in-human (FIH) single ascending dose (SAD) study for Lu AF82422, a human recombinant, anti-alpha-synuclein monoclonal antibody (mAb) in development for treatment of synucleinopathies. Alpha-synuclein is primarily expressed in brain, peripheral nerves and in blood cells. A tissue cross-reactivity assessment showed that Lu AF82422 binding was generally restricted to nervous tissues. Flow cytometry analysis did not show extracellular surface binding of Lu AF82422 to human platelets, erythrocytes, granulocytes, or lymphocytes, but to a low fraction of monocytes, without any functional consequences on activation or phagocytic capacity. A single dose pharmacokinetic (PK) study in cynomolgus monkeys with dose levels of 1-30 mg/kg confirmed PK properties in the expected range for a mAb with a soluble target, and target engagement was shown as a decrease in free alpha-synuclein in plasma. Four-week repeat-dose toxicity studies were conducted in rats and cynomolgus monkeys at doses up to 600 mg/kg administered intravenously every 10 days. Results showed no treatment-related adverse findings and the no-observed-adverse-effect-level was the highest dose tested. Target engagement was shown in plasma and cerebrospinal fluid. Taken together, the nonclinical data indicated no safety signal of concern and provided adequate safety margins between observed safe doses in animals and the planned dose levels in the FIH SAD study.

Encapsulated galanin-producing cells attenuate focal epileptic seizures in the hippocampus.

PURPOSE: Encapsulated cell biodelivery (ECB) is a relatively safe approach, since the devices can be removed in the event of adverse effects. The main objectives of the present study were to evaluate whether ECB could be a viable alternative of cell therapy for epilepsy. We therefore developed a human cell line producing galanin, a neuropeptide that has been shown to exert inhibitory effects on seizures, most likely acting via decreasing glutamate release from excitatory synapses. To explore whether ECB of genetically modified galanin-producing human cell line could provide seizure-suppressant effects, and test possible translational prospect for clinical application, we implanted ECB devices bilaterally into the hippocampus of rats subjected to rapid kindling, a model for recurrent temporal lobe seizures. METHODS: Two clones from a genetically modified human cell line secreting different levels of galanin were tested. Electroencephalography (EEG) recordings and stimulations were performed by electrodes implanted into the hippocampus at the same surgical session as ECB devices. One week after the surgery, rapid kindling stimulations were initiated. KEY FINDINGS: Enzyme-linked immunosorbent assay (ELISA) measurements prior to device implantation showed a release of galanin on average of 8.3 ng/mL/24 h per device for the low-releasing clone and 12.6 ng/mL/24 h per device for the high-releasing clone. High-releasing galanin-producing ECB devices moderately decreased stimulation-induced focal afterdischarge duration, whereas low-releasing ECB devices had no significant effect. SIGNIFICANCE: Our study shows that galanin-releasing ECB devices moderately suppress focal stimulation-induced recurrent seizures. Despite this moderate effect, the study provides conceptual proof that ECB could be a viable alternative approach to cell therapy in humans, with the advantage that the treatment could be terminated by removing these devices from the brain. Thereby, this strategy provides a higher level of safety for future therapeutic applications, in which genetically modified human cell lines that are optimized to produce and release antiepileptic compounds could be clinically evaluated for their seizure-suppressant effects.

Encapsulated cell-based biodelivery of meteorin is neuroprotective in the quinolinic acid rat model of neurodegenerative disease.

PURPOSE: Encapsulated cell (EC) biodelivery is a promising, clinically relevant technology platform to safely target the delivery of therapeutic proteins to the central nervous system. The purpose of this study was to evaluate EC biodelivery of the novel neurotrophic factor, Meteorin, to the striatum of rats and to investigate its neuroprotective effects against quinolinic acid (QA)-induced excitotoxicity. METHODS: Meteorin-producing ARPE-19 cells were loaded into EC biodelivery devices and implanted into the striatum of rats. Two weeks after implantation, QA was injected into the ipsilateral striatum followed by assessment of neurological performance two and four weeks after QA administration. RESULTS: Implant-delivered Meteorin effectively protected against QA-induced toxicity, as manifested by both near-normal neurological performance and reduction of brain cell death. Morphological analysis of the Meteorin-treated brains showed a markedly reduced striatal lesion size. The EC biodelivery devices produced stable or even increasing levels of Meteorin throughout the study over 6 weeks. CONCLUSIONS: Stereotactically implanted EC biodelivery devices releasing Meteorin could offer a feasible strategy in the treatment of neurological diseases with an excitotoxic component such as Huntington's disease. In a broader sense, the EC biodelivery technology is a promising therapeutic protein delivery platform for the treatment of a wide range of diseases of the central nervous system.

Cometin is a novel neurotrophic factor that promotes neurite outgrowth and neuroblast migration in vitro and supports survival of spiral ganglion neurons in vivo.

Neurotrophic factors are secreted proteins responsible for migration, growth and survival of neurons during development, and for maintenance and plasticity of adult neurons. Here we present a novel secreted protein named Cometin which together with Meteorin defines a new evolutionary conserved protein family. During early mouse development, Cometin is found exclusively in the floor plate and from E13.5 also in dorsal root ganglions and inner ear but apparently not in the adult nervous system. In vitro, Cometin promotes neurite outgrowth from dorsal root ganglion cells which can be blocked by inhibition of the Janus or MEK kinases. In this assay, additive effects of Cometin and Meteorin are observed indicating separate receptors. Furthermore, Cometin supports migration of neuroblasts from subventricular zone explants to the same extend as stromal cell derived factor 1a. Given the neurotrophic properties in vitro, combined with the restricted inner ear expression during development, we further investigated Cometin in relation to deafness. In neomycin deafened guinea pigs, two weeks intracochlear infusion of recombinant Cometin supports spiral ganglion neuron survival and function. In contrast to the control group receiving artificial perilymph, Cometin treated animals retain normal electrically-evoked brainstem response which is maintained several weeks after treatment cessation. Neuroprotection is also evident from stereological analysis of the spiral ganglion. Altogether, these studies show that Cometin is a potent new neurotrophic factor with therapeutic potential.

Lentiviral delivery of meteorin protects striatal neurons against excitotoxicity and reverses motor deficits in the quinolinic acid rat model.

Meteorin is a newly discovered secreted protein involved in both glial and neuronal cell differentiation, as well as in cerebral angiogenesis during development; but effects in the adult nervous system are unknown. The growth factor-like properties and expression of Meteorin during the development of the nervous system raises the possibility that it might possess important neuroprotective or regenerative capabilities. This report is the first demonstration that Meteorin has potent neuroprotective effects in vivo. Lentiviral-mediated striatal delivery of Meteorin to rats two weeks prior to injections of quinolinic acid (QA) dramatically reduced the loss of striatal neurons. The cellular protection afforded by Meteorin was associated with normalization of neurological performance on spontaneous forelimb placing and cylinder behavioral tests and a complete protection against QA-induced weight loss. These benefits were comparable in magnitude to those obtained with lentiviral-mediated delivery of ciliary neurotrophic factor (CNTF), a protein with known neuroprotective properties in the same model system. In naive animals, endogenous levels of both Meteorin and CNTF were increased in glial cells in response to QA lesion indicating that Meteorin may exert its protective effects as part of the reactive gliosis cascade in the injured brain. In summary, these data demonstrate that Meteorin strongly protects striatal neurons and deserves additional evaluation as a novel therapeutic for the treatment of neurological disorders with an excitotoxic component such as Huntington's Disease.

Long-term delivery of nerve growth factor by encapsulated cell biodelivery in the Göttingen minipig basal forebrain.

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

Characterization of Meteorin--an evolutionary conserved neurotrophic factor.

Growth factors control cellular growth, proliferation, and differentiation and may have therapeutic applications. In this study, we focus on Meteorin which is a member of a largely uncharacterized evolutionary conserved two-member growth factor family. Our analysis shows that Meteorin is expressed in the central nervous system both during development and in adult mice. Detailed immunohistological analysis of the adult mouse brain reveals that Meteorin is highly expressed in Bergmann glia and in a few discrete neuronal populations residing in the superior colliculus, the ocular motor nucleus, the raphe and pontine nuclei, and in various thalamic nuclei. In addition, low levels of Meteorin is found in astrocytes (S100beta+, OX42-) distributed ubiquitously throughout the brain. Meteorin was cloned and recombinant protein purified allowing N-terminal sequencing and mass spectrometric analysis showing that Meteorin is secreted as an unmodified monomer. This form is bioactive as it induces neurite outgrowth from dorsal root ganglions in vitro. Intrastriatal protein injection and lentiviral studies in vivo showed that Meteorin is a highly diffusible molecule in the brain and cellular uptake is apparent in specific populations which may carry the receptor. In summary, we provide a comprehensive expression analysis and have made and thoroughly validated molecular tools to help investigate the therapeutic potential of Meteorin.

GDNF released from encapsulated cells suppresses seizure activity in the epileptic hippocampus.

To date, a variety of pharmacological treatments exists for patients suffering epilepsy, but systemically administered drugs offer only symptomatic relief and often cause unwanted side effects. Moreover, available drugs are not effective in one third of the patients. Thus, more local and more effective treatment strategies need to be developed. Gene therapy-based expression of endogenous anti-epileptic agents represents a novel approach that could interfere with the disease process and result in stable and long-term suppression of seizures in epilepsy patients. We have reported earlier that direct in vivo viral vector-mediated overexpression of the glial cell line-derived neurotrophic factor (GDNF) in the rat hippocampus suppressed seizures in different animal models of epilepsy. Here we explored whether transplantation of encapsulated cells that release GDNF in the hippocampus could also exert a seizure-suppressant effect. Such ex vivo gene therapy approach represents a novel, more clinically safe approach, since the treatment could be terminated by retrieving the transplants from the brain. We demonstrate here that encapsulated cells, which are genetically modified to produce and release GDNF, can suppress recurrent generalized seizures when implanted into the hippocampus of kindled rats.

Midbrain expression of Delta-like 1 homologue is regulated by GDNF and is associated with dopaminergic differentiation.

Affymetrix GeneChip technology and quantitative real-time PCR (Q-PCR) were used to examine changes in gene expression in the adult murine substantia nigra pars compacta (SNc) following lentiviral glial cell line-derived neurotrophic factor (GDNF) delivery in adult striatum. We identified several genes that were upregulated after GDNF treatment. Among these, the gene encoding the transmembrane protein Delta-like 1 homologue (Dlk1) was upregulated with a greater than 4-fold increase in mRNA encoding this protein. Immunohistochemistry with a Dlk1-specific antibody confirmed the observed upregulation with increased positive staining of cell bodies in the SNc and fibers in the striatum. Analysis of the developmental regulation of Dlk1 in the murine ventral midbrain showed that the upregulation of Dlk1 mRNA correlated with the generation of tyrosine hydroxylase (TH)-positive neurons. Furthermore, Dlk1 expression was analyzed in MesC2.10 cells, which are derived from embryonic human mesencephalon and capable of undergoing differentiation into dopaminergic neurons. We detected upregulation of Dlk1 mRNA and protein under conditions where MesC2.10 cells differentiate into a dopaminergic phenotype (41.7+/-7.1% Dlk1+ cells). In contrast, control cultures subjected to default differentiation into non-dopaminergic neurons only expressed very few (3.7+/-1.3%) Dlk1-immunopositive cells. The expression of Dlk1 in MesC2.10 cells was specifically upregulated by the addition of GDNF. Thus, our data suggest that Dlk1 expression precedes the appearance of TH in mesencephalic cells and that levels of Dlk1 are regulated by GDNF.

Efficient in vivo protection of nigral dopaminergic neurons by lentiviral gene transfer of a modified Neurturin construct.

Protein injection studies of the glial cell line derived neurotrophic factor (GDNF) family member Neurturin (NTN) have demonstrated neuroprotective effects on dopaminergic (DA) neurons, which are selectively lost during Parkinson's disease (PD). However, unlike GDNF, NTN has not previously been applied in PD models using an in vivo gene therapy approach. Difficulties with lentiviral gene delivery of wild type (wt) NTN led us to examine the role of the pre-pro-sequence, and to evaluate different NTN constructs in order to optimize gene therapy with NTN. Results from transfected cultured cells showed that wt NTN was poorly processed, and secreted as a pro-form. A similarly poor processing was found with a chimeric protein consisting of the pre-pro-part from GDNF and mature NTN. Moreover, we found that the biological activity of pro-NTN differs from mature NTN, as pro-NTN did not form a signaling complex with the tyrosine kinase receptor Ret and GFRalpha2 or GFRalpha1. Deletion of the pro-region resulted in significantly higher secretion of active NTN, which was further increased when substituting the wt NTN signal peptide with the immunoglobulin heavy-chain signal peptide (IgSP). The enhanced secretion of active mature NTN using the IgSP-NTN construct was reproduced in vivo in lentiviral-transduced rat striatal cells and, unlike wt NTN, enabled efficient neuroprotection of lesioned nigral DA neurons, similar to GDNF. An in vivo gene therapy approach with a modified NTN construct is therefore a possible treatment option for Parkinson's disease that should be further explored.