RESUMEN
There are few treatments that slow neurodegeneration in Alzheimer's disease (AD), and while therapeutic antibodies are being investigated in clinical trials for AD treatment, their access to the central nervous system is restricted by the blood-brain barrier. This study investigates a bispecific modular fusion protein composed of gantenerumab, a fully human monoclonal anti- amyloid-beta (Aß) antibody under investigation for AD treatment, with a human transferrin receptor 1-directed Brainshuttle™ module (trontinemab; RG6102, INN trontinemab). In vitro, trontinemab showed a similar binding affinity to fibrillar Aß40 and Aß plaques in human AD brain sections to gantenerumab. A single intravenous administration of trontinemab (10 mg/kg) or gantenerumab (20 mg/kg) to non-human primates (NHPs, Macaca fascicularis), was well tolerated in both groups. Immunohistochemistry indicated increased trontinemab uptake into the brain endothelial cell layer and parenchyma, and more homogeneous distribution, compared with gantenerumab. Brain and plasma pharmacokinetic (PK) parameters for trontinemab were estimated by nonlinear mixed-effects modeling with correction for tissue residual blood, indicating a 4-18-fold increase in brain exposure. A previously developed clinical PK/pharmacodynamic model of gantenerumab was adapted to include a brain compartment as a driver of plaque removal and linked to the allometrically scaled above model from NHP. The new brain exposure-based model was used to predict trontinemab dosing regimens for effective amyloid reduction. Simulations from these models were used to inform dosing of trontinemab in the first-in-human clinical trial.
Asunto(s)
Enfermedad de Alzheimer , Anticuerpos Monoclonales , Animales , Humanos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/uso terapéutico , Anticuerpos Monoclonales/farmacología , Encéfalo/metabolismo , Primates/metabolismoRESUMEN
Mutations in glucocerebrosidase cause the lysosomal storage disorder Gaucher's disease and are the most common risk factor for Parkinson's disease. Therapies to restore the enzyme's function in the brain hold great promise for treating the neurological implications. Thus, we developed blood-brain barrier penetrant therapeutic molecules by fusing transferrin receptor-binding moieties to ß-glucocerebrosidase (referred to as GCase-BS). We demonstrate that these fusion proteins show significantly increased uptake and lysosomal efficiency compared to the enzyme alone. In a cellular disease model, GCase-BS rapidly rescues the lysosomal proteome and lipid accumulations beyond known substrates. In a mouse disease model, intravenous injection of GCase-BS leads to a sustained reduction of glucosylsphingosine and can lower neurofilament-light chain plasma levels. Collectively, these findings demonstrate the potential of GCase-BS for treating GBA1-associated lysosomal dysfunction, provide insight into candidate biomarkers, and may ultimately open a promising treatment paradigm for lysosomal storage diseases extending beyond the central nervous system.
Asunto(s)
Enfermedad de Gaucher , Enfermedad de Parkinson , Animales , Ratones , Enfermedad de Gaucher/genética , Glucosilceramidasa/genética , Glucosilceramidasa/metabolismo , Encéfalo/metabolismo , Neuronas/metabolismo , Enfermedad de Parkinson/metabolismo , Lisosomas/metabolismo , Mutación , alfa-Sinucleína/metabolismoRESUMEN
Esophageal cancer is associated with a dismal prognosis. The armamentarium of approved drugs is focused on chemotherapy with modest therapeutic benefit. Recently, checkpoint inhibitory monoclonal antibody Pembrolizumab was approved. In order to identify new targets and modalities for the treatment of esophagus squamous cell carcinoma (ESCC) we searched the literature for circRNAs involved in the pathogenesis of ESCC. We identified two down-regulated and 17 up-regulated circRNAs as well as a synthetic circRNA with efficacy in preclinical in vivo systems. Down-regulated circRNAs sponge microRNAs directed against tumor suppressor genes. Up-regulated circRNAs sponge microRNAs directed against mRNAs, which encode proteins with pro-tumoral functions. We discuss issues such as reconstitution of down-regulated circRNAs and inhibition of up-regulated circRNAs with short interfering RNA (siRNA)- related entities. Also, we address druggability issues of the identified targets.
Asunto(s)
Neoplasias Esofágicas , Carcinoma de Células Escamosas de Esófago , MicroARNs , Línea Celular Tumoral , Neoplasias Esofágicas/tratamiento farmacológico , Neoplasias Esofágicas/genética , Neoplasias Esofágicas/patología , Carcinoma de Células Escamosas de Esófago/tratamiento farmacológico , Carcinoma de Células Escamosas de Esófago/genética , Carcinoma de Células Escamosas de Esófago/patología , Regulación Neoplásica de la Expresión Génica , Humanos , MicroARNs/genética , MicroARNs/metabolismo , ARN Circular/genéticaRESUMEN
BACKGROUND: The pathways that control protein transport across the blood-brain barrier (BBB) remain poorly characterized. Despite great advances in recapitulating the human BBB in vitro, current models are not suitable for systematic analysis of the molecular mechanisms of antibody transport. The gaps in our mechanistic understanding of antibody transcytosis hinder new therapeutic delivery strategy development. METHODS: We applied a novel bioengineering approach to generate human BBB organoids by the self-assembly of astrocytes, pericytes and brain endothelial cells with unprecedented throughput and reproducibility using micro patterned hydrogels. We designed a semi-automated and scalable imaging assay to measure receptor-mediated transcytosis of antibodies. Finally, we developed a workflow to use CRISPR/Cas9 gene editing in BBB organoid arrays to knock out regulators of endocytosis specifically in brain endothelial cells in order to dissect the molecular mechanisms of receptor-mediated transcytosis. RESULTS: BBB organoid arrays allowed the simultaneous growth of more than 3000 homogenous organoids per individual experiment in a highly reproducible manner. BBB organoid arrays showed low permeability to macromolecules and prevented transport of human non-targeting antibodies. In contrast, a monovalent antibody targeting the human transferrin receptor underwent dose- and time-dependent transcytosis in organoids. Using CRISPR/Cas9 gene editing in BBB organoid arrays, we showed that clathrin, but not caveolin, is required for transferrin receptor-dependent transcytosis. CONCLUSIONS: Human BBB organoid arrays are a robust high-throughput platform that can be used to discover new mechanisms of receptor-mediated antibody transcytosis. The implementation of this platform during early stages of drug discovery can accelerate the development of new brain delivery technologies.
Asunto(s)
Anticuerpos/metabolismo , Bioingeniería/métodos , Barrera Hematoencefálica/metabolismo , Organoides/metabolismo , Receptores de Transferrina/metabolismo , Transcitosis/fisiología , Animales , Anticuerpos/análisis , Astrocitos/química , Astrocitos/metabolismo , Barrera Hematoencefálica/química , Barrera Hematoencefálica/citología , Células Cultivadas , Técnicas de Cocultivo , Células Endoteliales/química , Células Endoteliales/metabolismo , Humanos , Organoides/química , Organoides/citología , Pericitos/química , Pericitos/metabolismo , Receptores de Transferrina/análisisRESUMEN
Reducing Amyloid ß (Aß) in the brain is of fundamental importance for advancing the therapeutics for Alzheimer`s disease. The endogenous metallopeptidase neprilysin (NEP) has been identified as one of the key Aß-degrading enzymes. Delivery of NEP to the brain by utilizing the Brain Shuttle (BS) transport system offers a promising approach for clearing central Aß. We fused the extracellular catalytic domain of NEP to an active or inactive BS module. The two BS-NEP constructs were used to investigate the pharmacokinetic/pharmacodynamics relationships in the blood and the cerebrospinal fluid (CSF) in dose-response and multiple dosing. As previously shown, NEP was highly effective at degrading Aß in blood but not in the CSF compartment after systemic administration. In contrast, the NEP with an active BS module led to a significant CSF exposure of BS-NEP, followed by substantial Aß reduction in CSF and brain parenchyma. Our data show that a BS module against the transferrin receptor facilitates the transport of an Aß degrading enzyme across the blood-brain barriers to efficiently reduce Aß levels in both CSF and brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/antagonistas & inhibidores , Encéfalo/metabolismo , Neprilisina/farmacología , Proteínas Recombinantes de Fusión/farmacología , Péptidos beta-Amiloides/deficiencia , Animales , Barrera Hematoencefálica/metabolismo , Células HEK293 , Humanos , Neprilisina/líquido cefalorraquídeo , Neprilisina/farmacocinética , Ratas , Ratas Wistar , Proteínas Recombinantes de Fusión/líquido cefalorraquídeo , Proteínas Recombinantes de Fusión/farmacocinéticaRESUMEN
PURPOSE: Administration of therapeutic monoclonal antibodies (mAbs) is frequently accompanied by severe first infusion reactions (FIR). The mechanism driving FIR is still unclear. This study aimed to investigate the cellular and molecular mechanisms causing FIR in humanized mouse models and their potential for evaluating FIR risk in patients. METHODS: Mice humanized for Fc gamma receptors (FcγRs) were generated by recombination-mediated genomic replacement. Body temperature, cytokine release and reactive oxygen species (ROS) were measured to assess FIR to mAbs. RESULTS: Infusion of human mAb specific for mouse transferrin receptor (HamTfR) into FcγR-humanized mice, produced marked transient hypothermia accompanied by an increase in inflammatory cytokines KC and MIP-2, and ROS. FIR were dependent on administration route and Fc-triggered effector functions mediated by neutrophils. Human neutrophils also induced FIR in wild type mice infused with HamTfR. Specific knock-in mice demonstrated that human FcγRIIIb on neutrophils was both necessary and sufficient to cause FIR. FcγRIIIb-mediated FIR was abolished by depleting neutrophils or blocking FcγRIIIb with CD11b antibodies. CONCLUSIONS: Human FcγRIIIb and neutrophils are primarily responsible for triggering FIR. Clinical strategies to prevent FIR in patients should focus on this pathway and may include transient depletion of neutrophils or blocking FcγRIIIb with specific mAbs.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Hipotermia/inducido químicamente , Inflamación/inducido químicamente , Neutrófilos/inmunología , Receptores de IgG/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/inmunología , Humanos , Hipotermia/inmunología , Inflamación/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neutrófilos/efectos de los fármacos , Receptores de IgG/genética , Receptores de Transferrina/inmunologíaRESUMEN
Receptors show promise for the transport of monoclonal antibodies (mAbs) across the blood-brain barrier. However, safety liabilities associated with peripheral receptor binding and Fc effector function have been reported. We present the Brain Shuttle-mAb (BS-mAb) technology, and we investigate the role of Fc effector function in vitro and in an Fcγ receptor (FcγR)-humanized mouse model. Strong first infusion reactions (FIRs) were observed for a conventional mAb against transferrin receptor (TfR) with a wild-type immunoglobulin G1 (IgG1) Fc. Fc effector-dead constructs completely eliminated all FIRs. Remarkably, no FIR was observed for the BS-mAb construct with a native IgG1 Fc function. Using various BS-mAb constructs, we show that TfR binding through the C-terminal BS module attenuates Fc-FcγR interactions, primarily because of steric hindrance. Nevertheless, BS-mAbs maintain effector function activity when binding their brain target. Thus, mAbs with full effector function can be transported in a stealth mode in the periphery while fully active when engaged with their brain target.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Anticuerpos Monoclonales , Barrera Hematoencefálica/metabolismo , Sistemas de Liberación de Medicamentos , Inmunoglobulina G/farmacología , Receptores de IgG/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Barrera Hematoencefálica/patología , Células CHO , Cricetulus , Humanos , Masculino , Ratones , Ratones Transgénicos , Receptores de IgG/genéticaRESUMEN
Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidß (Aß) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aß levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aß. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Encéfalo/metabolismo , Fragmentos de Péptidos/administración & dosificación , Administración Intravenosa , Secretasas de la Proteína Precursora del Amiloide/química , Animales , Ácido Aspártico Endopeptidasas/química , Barrera Hematoencefálica/metabolismo , Dominio Catalítico/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Humanos , Ratones , Fragmentos de Péptidos/farmacología , Receptores de Transferrina/metabolismoRESUMEN
We have adapted an in vitro model of the human blood-brain barrier, the immortalized human cerebral microvascular endothelial cells (hCMEC/D3), to quantitatively measure protein transcytosis. After validating the receptor-mediated transport using transferrin, the system was used to measure transcytosis rates of antibodies directed against potential brain shuttle receptors. While an antibody to the insulin-like growth factor 1 receptor (IGF1R) was exclusively recycled to the apical compartment, the fate of antibodies to the transferrin receptor (TfR) was determined by their relative affinities at extracellular and endosomal pH. An antibody with reduced affinity at pH5.5 showed significant transcytosis, while pH-independent antibodies of comparable affinities at pH 7.4 remained associated with intracellular vesicular compartments and were finally targeted for degradation.
Asunto(s)
Anticuerpos/metabolismo , Barrera Hematoencefálica/metabolismo , Transcitosis , Anticuerpos/inmunología , Antígenos CD/inmunología , Línea Celular , Humanos , Concentración de Iones de Hidrógeno , Receptor IGF Tipo 1/inmunología , Receptores de Transferrina/inmunologíaRESUMEN
Although biotherapeutics have vast potential for treating brain disorders, their use has been limited due to low exposure across the blood-brain barrier (BBB). We report that by manipulating the binding mode of an antibody fragment to the transferrin receptor (TfR), we have developed a Brain Shuttle module, which can be engineered into a standard therapeutic antibody for successful BBB transcytosis. Brain Shuttle version of an anti-Aß antibody, which uses a monovalent binding mode to the TfR, increases ß-Amyloid target engagement in a mouse model of Alzheimer's disease by 55-fold compared to the parent antibody. We provide in vitro and in vivo evidence that the monovalent binding mode facilitates transcellular transport, whereas a bivalent binding mode leads to lysosome sorting. Enhanced target engagement of the Brain Shuttle module translates into a significant improvement in amyloid reduction. These findings have major implications for the development of biologics-based treatment of brain disorders.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Transporte de Proteínas/fisiología , Anticuerpos de Cadena Única/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/genética , Animales , Barrera Hematoencefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/inmunología , Línea Celular Transformada , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Macrólidos/farmacología , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Presenilina-1/genética , Unión Proteica/efectos de los fármacos , Unión Proteica/inmunología , Transporte de Proteínas/efectos de los fármacos , Receptores de Transferrina/inmunología , Receptores de Transferrina/metabolismo , Anticuerpos de Cadena Única/farmacología , Anticuerpos de Cadena Única/uso terapéutico , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Factores de Tiempo , Transcitosis/efectos de los fármacos , Transcitosis/genética , Transcitosis/inmunologíaRESUMEN
BACKGROUND: Thymic stromal lymphopoietin (TSLP) pathway blockade is a potential strategy for asthma treatment because the main activities of TSLP are activation of myeloid dendritic cells (mDCs) and modulation of cytokine production by mast cells. TSLP-activated mDCs prime the differentiation of naive T cells into inflammatory TH2 cells. OBJECTIVE: We sought to investigate mechanisms underlying the development of allergic lung inflammation in cynomolgus monkeys using gene expression profiling and to assess the effect of thymic stromal lymphopoietin receptor (TSLPR) blockade in this model. METHODS: An mAb against human TSLPR was generated and confirmed to be cross-reactive to cynomolgus monkey. Animals were dosed weekly with either vehicle or anti-TSLPR mAb for 6 weeks, and their responses to allergen challenge at baseline, week 2, and week 6 were assessed. RESULTS: After 6 weeks of treatment, anti-TSLPR mAb-treated animals showed reduced bronchoalveolar lavage (BAL) fluid eosinophil counts, reduced airway resistance in response to allergen challenge, and reduced IL-13 cytokine levels in BAL fluid compared with values seen in vehicle-treated animals. Expression profiling of BAL fluid cells collected before and after challenge showed a group of genes upregulated by allergen challenge that strongly overlapped with 11 genes upregulated in dendritic cells (DCs) when in vitro stimulated by TSLP (TSLP-DC gene signature). The number of genes differentially expressed in response to challenge was reduced in antibody-treated animals after 6 weeks relative to vehicle-treated animals. Expression of the TSLP-DC gene signature was also significantly reduced in antibody-treated animals. CONCLUSION: These results demonstrate promising efficacy for TSLPR blockade in an allergic lung inflammation model in which TSLP activation of mDCs might play a key role.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Asma/terapia , Modelos Animales de Enfermedad , Hipersensibilidad/terapia , Inflamación/terapia , Receptores de Citocinas/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Asma/inmunología , Cricetinae , Citocinas/inmunología , Citocinas/metabolismo , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Femenino , Humanos , Hipersensibilidad/inmunología , Inflamación/inmunología , Macaca fascicularis/inmunología , Receptores de Citocinas/inmunología , Células Th2/inmunología , Células Th2/metabolismo , Linfopoyetina del Estroma TímicoRESUMEN
The tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a multifunctional cytokine playing a key role in tissue regeneration and remodeling. Dysregulation of TWEAK signaling is involved in various pathological processes like autoimmune diseases and cancer. The unique interaction with its cognate receptor Fn14 makes both ligand and receptor promising targets for novel therapeutics. To gain insights into this important signaling pathway, we determined the structure of soluble human TWEAK in complex with the Fab fragment of an antibody selected for inhibition of receptor binding. In the crystallized complex TWEAK is bound by three Fab fragments of the neutralizing antibody. Homology modeling shows that Fab binding overlaps with the putative Fn14 binding site of TWEAK. Docking of the Fn14 cysteine rich domain (CRD) to that site generates a highly complementary interface with perfectly opposing charged and hydrophobic residues. Taken together the presented structure provides new insights into the biology of TWEAK and the TWEAK/Fn14 pathway, which will help to optimize the therapeutic strategy for treatment of related cancer types and autoimmune diseases.
Asunto(s)
Anticuerpos Neutralizantes/química , Fragmentos Fab de Inmunoglobulinas/química , Modelos Moleculares , Conformación Proteica , Factores de Necrosis Tumoral/química , Cristalografía , Citocina TWEAK , Humanos , Fragmentos Fab de Inmunoglobulinas/metabolismo , Unión Proteica , Factores de Necrosis Tumoral/metabolismoRESUMEN
BACKGROUND: The aim of this study was to characterize the hCMEC/D3 cell line, an in vitro model of the human Blood Brain Barrier (BBB) for the expression of brain endothelial specific claudins-3 and -12. FINDINGS: hCMEC/D3 cells express claudins-3 and -12. Claudin-3 is distinctly localized to the TJ whereas claudin -12 is observed in the perinuclear region and completely absent from TJs. We show that the expression of both proteins is lost in cell passage numbers where the BBB properties are no longer fully conserved. Expression and localization of claudin-3 is not modulated by simvastatin shown to improve barrier function in vitro and also recommended for routine hCMEC/D3 culture. CONCLUSIONS: These results support conservation of claudin-3 and -12 expression in the hCMEC/D3 cell line and make claudin-3 a potential marker for BBB characteristics in vitro.