RESUMEN
Monoclonal antibodies (mAbs) have high binding specificity and affinity, making them attractive for treating brain diseases. However, their effectiveness is limited by poor blood-brain barrier (BBB) penetration and rapid central nervous system (CNS) clearance. Our group identified blood-brain barrier modulator (BBBM) peptides that improved mAb penetration across the BBB into the brain. In this study, we investigated the pharmacokinetics of a mAb delivered to the brain using BBBMs after intravenous (IV) administration and explored the impact of antibody format (size, neonatal Fc receptor (FcRn) binding, hyaluronic acid binding) on brain clearance following direct injection into the central nervous system (CNS) via intracerebroventricular (ICV) injection. IRDye800CW-labeled antibodies were administered into C57BL/6 mice via ICV or IV injection, and organ concentrations were measured after various time points. When a mAb was coadministered with a BBBM peptide, the permeation of mAb across the BBB was increased compared to mAb alone at early time points; however, the mAb was cleared within 2 h from the brain. ICV experiments revealed that an antibody Fab fragment had a higher brain exposure than a mAb, and that a Fab fused to a hyaluronic acid binding domain (Fab-VG1) showed remarkable improvement in brain exposure. These findings suggest that BBBMs and antibody format optimization may be promising strategies for enhancing brain retention of therapeutic antibodies.
RESUMEN
Monoclonal antibodies (mAbs) possess favorable pharmacokinetic properties, high binding specificity and affinity, and minimal off-target effects, making them promising therapeutic agents for central nervous system (CNS) disorders. However, their development as effective therapeutic and diagnostic agents for brain disorders is hindered by their limited ability to efficiently penetrate the blood-brain barrier (BBB). Therefore, it is crucial to develop efficient delivery methods that enhance the penetration of antibodies into the brain. Previous studies have demonstrated the potential of cadherin-derived peptides (i.e., ADTC5, HAVN1 peptides) as BBB modulators (BBBMs) to increase paracellular porosities for penetration of molecules across the BBB. Here, we test the effectiveness of the leading BBBM peptide, HAVN1 (Cyclo(1,6)SHAVSS), in enhancing the permeation of various monoclonal antibodies through the BBB using both in vitro and in vivo systems. In vitro, HAVN1 has been shown to increase the permeability of fluorescently labeled macromolecules, such as a 70 kDa dextran, 50 kDa Fab1, and 150 kDa mAb1, by 4- to 9-fold in a three-dimensional blood-brain barrier (3D-BBB) microfluidics model using a human BBB endothelial cell line (i.e., hCMEC/D3). HAVN1 was selective in modulating the BBB endothelial cell, compared to the pulmonary vascular endothelial (PVE) cell barrier. Co-administration of HAVN1 significantly improved brain depositions of mAb1, mAb2, and Fab1 in C57BL/6 mice after 15 min in the systemic circulation. Furthermore, HAVN1 still significantly enhanced brain deposition of mAb2 when it was administered 24 h after the administration of the mAb. Lastly, we observed that multiple doses of HAVN1 may have a cumulative effect on the brain deposition of mAb2 within a 24-h period. These findings offer promising insights into optimizing HAVN1 and mAb dosing regimens to control or modulate mAb brain deposition for achieving desired mAb dose in the brain to provide its therapeutic effects.
Asunto(s)
Barrera Hematoencefálica , Microfluídica , Ratones , Animales , Humanos , Barrera Hematoencefálica/metabolismo , Ratones Endogámicos C57BL , Encéfalo/metabolismo , Péptidos/metabolismo , Modelos Animales , Anticuerpos Monoclonales/metabolismoRESUMEN
Over the past few decades, the complexity of molecular entities being advanced for therapeutic purposes has continued to evolve. A main propellent fueling innovation is the perpetual mandate within the pharmaceutical industry to meet the needs of novel disease areas and/or delivery challenges. As new mechanisms of action are uncovered, and as our understanding of existing mechanisms grows, the properties that are required and/or leveraged to enable therapeutic development continue to expand. One rapidly evolving area of interest is that of chemically enhanced peptide and protein therapeutics. While a variety of conjugate molecules such as antibody-drug conjugates, peptide/protein-PEG conjugates, and protein conjugate vaccines are already well established, others, such as antibody-oligonucleotide conjugates and peptide/protein conjugates using non-PEG polymers, are newer to clinical development. This review will evaluate the current development landscape of protein-based chemical conjugates with special attention to considerations such as modulation of pharmacokinetics, safety/tolerability, and entry into difficult to access targets, as well as bioavailability. Furthermore, for the purpose of this review, the types of molecules discussed are divided into two categories: (1) therapeutics that are enhanced by protein or peptide bioconjugation, and (2) protein and peptide therapeutics that require chemical modifications. Overall, the breadth of novel peptide- or protein-based therapeutics moving through the pipeline each year supports a path forward for the pursuit of even more complex therapeutic strategies.
RESUMEN
Treatment of age-related macular degeneration (AMD) with anti-vascular endothelial growth factor (VEGF) biologic agents has been shown to restore and maintain visual acuity for many patients afflicted with wet AMD. These agents are usually administered via intravitreal injection at a dosing interval of 4-8 weeks. Employment of long-acting delivery (LAD) technologies could improve the therapeutic outcome, ensure timely treatment, and reduce burden on patients, caregivers, and the health care system. Development of LAD approaches requires thorough testing in pre-clinical species; however, therapeutic proteins of human origin may not be well tolerated during testing in non-human species due to immunogenicity. Here, we have engineered a surrogate porcine antibody Fab fragment (pigG6.31) from a human antibody for testing ocular LAD technologies in a porcine model. The engineered Fab retains the VEGF-A-binding and inhibition properties of the parental human Fab and has stability properties suitable for LAD evaluation. Upon intravitreal injection in minipigs, pigG6.31 showed first-order clearance from the ocular compartments with vitreal elimination rates consistent with other molecules of this size. Application of the surrogate molecule in an in vivo evaluation in minipigs of a prototype of the port delivery (PD) platform indicated continuous ocular delivery from the implant, with release kinetics consistent with both the results from in vitro release studies and the efficacy observed in human clinical studies of the PD system with ranibizumab (PDS). Anti-drug antibodies in the serum against pigG6.31 were not detected over exposure durations up to 16 weeks, suggesting that this molecule has low porcine immunogenicity.
Asunto(s)
Inhibidores de la Angiogénesis , Degeneración Macular Húmeda , Animales , Humanos , Fragmentos Fab de Inmunoglobulinas/uso terapéutico , Inyecciones Intravítreas , Ingeniería de Proteínas , Ranibizumab/uso terapéutico , Porcinos , Porcinos Enanos/metabolismo , Tecnología , Factor A de Crecimiento Endotelial Vascular/metabolismo , Degeneración Macular Húmeda/tratamiento farmacológicoRESUMEN
Long-acting delivery (LAD) of ocular therapeutics has potential to improve the standard of care for ocular diseases, such as age-related macular degeneration (AMD), by increasing patient compliance and reducing overall treatment burden on patients and healthcare providers. Although relatively few ocular LAD technologies are currently on the market, a variety of emergent and novel protein engineering-based technologies are being investigated in both the laboratory and clinical settings. Here, we review some of the key indications and treatments that would benefit from the development of LAD for the treatment of ocular diseases and examine the current state of LAD technologies that leverage protein-engineering approaches as well as nascent technologies with potential for future impact.
Asunto(s)
Preparaciones de Acción Retardada/uso terapéutico , Ojo/efectos de los fármacos , Degeneración Macular/tratamiento farmacológico , Soluciones Oftálmicas/uso terapéutico , Proteínas/uso terapéutico , Sistemas de Liberación de Medicamentos/métodos , HumanosRESUMEN
The neonatal Fc receptor (FcRn) plays an important and well-known role in antibody recycling in endothelial and hematopoietic cells and thus it influences the systemic pharmacokinetics (PK) of immunoglobulin G (IgG). However, considerably less is known about FcRn's role in the metabolism of IgG within individual tissues after intravenous administration. To elucidate the organ distribution and gain insight into the metabolism of humanized IgG1 antibodies with different binding affinities FcRn, comparative biodistribution studies in normal CD-1 mice were conducted. Here, we generated variants of herpes simplex virus glycoprotein D-specific antibody (humanized anti-gD) with increased and decreased FcRn binding affinity by genetic engineering without affecting antigen specificity. These antibodies were expressed in Chinese hamster ovary cell lines, purified and paired radiolabeled with iodine-125 and indium-111. Equal amounts of I-125-labeled and In-111-labeled antibodies were mixed and intravenously administered into mice at 5 mg/kg. This approach allowed us to measure both the real-time IgG uptake (I-125) and cumulative uptake of IgG and catabolites (In-111) in individual tissues up to 1 week post-injection. The PK and distribution of the wild-type IgG and the variant with enhanced binding for FcRn were largely similar to each other, but vastly different for the rapidly cleared low-FcRn-binding variant. Uptake in individual tissues varied across time, FcRn binding affinity, and radiolabeling method. The liver and spleen emerged as the most concentrated sites of IgG catabolism in the absence of FcRn protection. These data provide an increased understanding of FcRn's role in antibody PK and catabolism at the tissue level.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Receptores Fc/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/genética , Células CHO , Cricetinae , Cricetulus , Femenino , Variación Genética , Humanos , Inmunoglobulina G/administración & dosificación , Inmunoglobulina G/genética , Inmunoglobulina G/metabolismo , Radioisótopos de Indio/administración & dosificación , Radioisótopos de Indio/farmacocinética , Radioisótopos de Yodo/administración & dosificación , Radioisótopos de Yodo/farmacocinética , Ratones , Proteolisis , Receptores Fc/genética , Distribución TisularRESUMEN
Subcutaneous (SC) delivery is a common route of administration for therapeutic monoclonal antibodies (mAbs) with pharmacokinetic (PK)/pharmacodynamic (PD) properties requiring long-term or frequent drug administration. An ideal in vivo preclinical model for predicting human PK following SC administration may be one in which the skin and overall physiological characteristics are similar to that of humans. In this study, the PK properties of a series of therapeutic mAbs following intravenous (IV) and SC administration in Göttingen minipigs were compared with data obtained previously from humans. The present studies demonstrated: (1) minipig is predictive of human linear clearance; (2) the SC bioavailabilities in minipigs are weakly correlated with those in human; (3) minipig mAb SC absorption rates are generally higher than those in human and (4) the SC bioavailability appears to correlate with systemic clearance in minipigs. Given the important role of the neonatal Fc-receptor (FcRn) in the PK of mAbs, the in vitro binding affinities of these IgGs against porcine, human and cynomolgus monkey FcRn were tested. The result showed comparable FcRn binding affinities across species. Further, mAbs with higher isoelectric point tended to have faster systemic clearance and lower SC bioavailability in both minipig and human. Taken together, these data lend increased support for the use of the minipig as an alternative predictive model for human IV and SC PK of mAbs.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales/farmacocinética , Modelos Inmunológicos , Administración Intravenosa , Animales , Anticuerpos Monoclonales/inmunología , Femenino , Humanos , Inyecciones Subcutáneas , Masculino , Porcinos , Porcinos EnanosRESUMEN
IgG was traditionally thought to neutralize virions by blocking their attachment to or penetration into mucosal epithelial cells, a common site of exposure to viruses. However, we describe an intracellular neutralizing action for an influenza hemagglutinin-specific monoclonal antibody, Y8-10C2 (Y8), which has neutralizing activity only at an acidic pH. When Y8 was applied to the basolateral surface of Madin-Darby canine kidney cells expressing the rat neonatal Fc receptor for IgG (FcRn), it significantly reduced viral replication following apical exposure of the cell monolayer to influenza virus. Virus neutralization by Y8 mAb was dependent on FcRn expression and its transport of IgG. As both FcRn and Y8 mAb bind their partners only at acidic pH, the Y8 mAb is proposed to carry out its antiviral activity intracellularly. Furthermore, the virus, Y8 mAb, and FcRn colocalized within endosomes, possibly inhibiting the fusion of viral envelopes with endosomal membranes during primary uncoating, and preventing the accumulation of the neutralized viral nucleoprotein antigen in the nucleus. Prophylactic administration of Y8 mAb before viral challenge in WT mice, but not FcRn-KO mice, conferred protection from lethality, prevented weight loss, resulted in a significant reduction in pulmonary virus titers, and largely reduced virus-induced lung pathology. Thus, this study reveals an intracellular mechanism for viral neutralization in polarized epithelial cells that is dependent on FcRn-mediated transport of neutralizing IgG.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Inmunoglobulina G/metabolismo , Infecciones por Orthomyxoviridae/inmunología , Receptores Fc/fisiología , Animales , Animales Recién Nacidos , Perros , Ratones , Ratones Noqueados , Orthomyxoviridae/inmunología , Ratas , Receptores Fc/genética , Receptores Fc/inmunologíaRESUMEN
Antibody pharmacokinetics and pharmacodynamics are often governed by biological processes such as binding to antigens and other cognate receptors. Emphasis must also be placed, however, on fundamental physicochemical properties that define antibodies as complex macromolecules, including shape, size, hydrophobicity, and charge. Electrostatic interactions between anionic cell membranes and the predominantly positive surface charge of most antibodies can influence blood concentration and tissue disposition kinetics in a manner that is independent of antigen recognition. In this context, the deliberate modification of antibodies by chemical means has been exploited as a valuable preclinical research tool to investigate the relationship between net molecular charge and biological disposition. Findings from these exploratory investigations may be summarized as follows: (I) shifts in isoelectric point of approximately one pI unit or more can produce measurable changes in tissue distribution and kinetics, (II) increases in net positive charge generally result in increased tissue retention and increased blood clearance, and (III) decreases in net positive charge generally result in decreased tissue retention and increased whole body clearance. Understanding electrostatic interactions between antibodies and biological matrices holds relevance in biotechnology, especially with regard to the development of immunoconjugates. The guiding principles and knowledge gained from preclinical evaluation of chemically modified antibodies will be discussed and placed in the context of therapeutic antibodies that are currently marketed or under development, with a particular emphasis on pharmacokinetic and disposition properties.
Asunto(s)
Anticuerpos/química , Anticuerpos/metabolismo , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Animales , Aniones/metabolismo , Anticuerpos/inmunología , Anticuerpos/farmacología , Antígenos/inmunología , Cationes/metabolismo , Membrana Celular/inmunología , Membrana Celular/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunoconjugados/inmunología , Inmunoconjugados/metabolismo , Punto Isoeléctrico , Ratones , Modelos Moleculares , Terapia Molecular Dirigida , Neoplasias/inmunología , Neoplasias/terapia , Unión Proteica , Ingeniería de Proteínas/métodos , Ratas , Electricidad Estática , Distribución Tisular/inmunologíaRESUMEN
Protein delivery across polarized epithelia is controlled by receptor-mediated transcytosis. Many studies have examined basolateral-to-apical trafficking of polymeric IgA (pIgA) by the polymeric immunoglobulin receptor (pIgR). Less is known about apical-to-basolateral transcytosis, the direction the neonatal Fc receptor (FcRn) transports maternal IgGs across intestinal epithelia. To compare apical-to-basolateral and basolateral-to-apical transcytosis, we co-expressed FcRn and pIgR in Madin-Darby canine kidney (MDCK) cells and used pulse-chase experiments with confocal microscopy to examine transport of apically applied IgG Fcgamma and basolaterally applied pIgA. Fcgamma and pIgA trafficking routes were initially separate but intermixed at later chase times. Fcgamma was first localized near the apical surface, but became more equally distributed across the cell, consistent with concomitant transcytosis and recycling. By contrast, pIgA transport was strongly unidirectional: pIgA shifted from near the basolateral surface to an apical location with increasing time. Some Fcgamma and pIgA fluorescence colocalized in early (EEA1-positive), recycling (Rab11a-positive), and transferrin (Tf)-positive common/basolateral recycling endosomes. Fcgamma became more enriched in Tf-positive endosomes with time, whereas pIgA was sorted from these compartments. Live-cell imaging revealed that vesicles containing Fcgamma or pIgA shared similar mobility characteristics and were equivalently affected by depolymerizing microtubules, indicating that both trafficking routes depended to roughly the same extent on intact microtubules.
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Inmunoglobulina A/metabolismo , Receptores Fc/metabolismo , Receptores de Inmunoglobulina Polimérica/metabolismo , Animales , Células Cultivadas , Perros , Endosomas/metabolismo , Microscopía Confocal , Microscopía Fluorescente , Transporte de ProteínasRESUMEN
Recent advances in imaging techniques along with more powerful in vitro and in vivo models of receptor-mediated ligand transport are facilitating advances in our understanding of how cells efficiently direct receptors and their cargo to target destinations within the cytoplasm and at the plasma membrane. Specifically, light and 3D electron microscopy studies examining the trafficking behavior of the neonatal Fc receptor (FcRn), a transport receptor for immunoglobulin G (IgG), have given us new insights into the dynamic interplay between the structural components of the cytosolic trafficking machinery, its protein regulators, and the receptors it directs to various locations within the cell. These studies build upon previous biochemical characterizations of FcRn transport and are allowing us to begin formulation of a more complete model for the intracellular trafficking of receptor-ligand complexes.
Asunto(s)
Inmunoglobulina G/metabolismo , Receptores Fc/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Humanos , Modelos Biológicos , Datos de Secuencia MolecularRESUMEN
In mammals the transfer of passive immunity from mother to young is mediated by the MHC-related receptor FcRn, which transports maternal IgG across epithelial cell barriers. In birds, maternal IgY in egg yolk is transferred across the yolk sac to passively immunize chicks during gestation and early independent life. The chicken yolk sac IgY receptor (FcRY) is the ortholog of the mammalian phospholipase A2 receptor, a mannose receptor family member, rather than an FcRn or MHC homolog. FcRn and FcRY both exhibit ligand binding at the acidic pH of endosomes and ligand release at the slightly basic pH of blood. Here we show that FcRY expressed in polarized mammalian epithelial cells functioned in endocytosis, bidirectional transcytosis, and recycling of chicken FcY/IgY. Confocal immunofluorescence studies demonstrated that IgY binding and endocytosis occurred at acidic but not basic pH, mimicking pH-dependent uptake of IgG by FcRn. Colocalization studies showed FcRY-mediated internalization via clathrin-coated pits and transport involving early and recycling endosomes. Disruption of microtubules partially inhibited apical-to-basolateral and basolateral-to-apical transcytosis, but not recycling, suggesting the use of different trafficking machinery. Our results represent the first cell biological evidence of functional equivalence between FcRY and FcRn and provide an intriguing example of how evolution can give rise to systems in which similar biological requirements in different species are satisfied utilizing distinct protein folds.
Asunto(s)
Inmunoglobulinas/metabolismo , Lectinas Tipo C/metabolismo , Lectinas de Unión a Manosa/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Fc/metabolismo , Saco Vitelino/inmunología , Animales , Línea Celular , Polaridad Celular , Embrión de Pollo , Endocitosis/inmunología , Células Epiteliales/citología , Células Epiteliales/inmunología , Células Epiteliales/metabolismo , Femenino , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/metabolismo , Inmunización Pasiva , Túbulos Renales Colectores/citología , Túbulos Renales Colectores/inmunología , Túbulos Renales Colectores/metabolismo , Lectinas Tipo C/genética , Receptor de Manosa , Lectinas de Unión a Manosa/genética , Ratas , Receptores de Superficie Celular/genética , Receptores Fc/genética , Receptores de Fosfolipasa A2/genética , Receptores de Fosfolipasa A2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transfección , Saco Vitelino/metabolismoRESUMEN
We have developed methods to locate individual ligands that can be used for electron microscopy studies of dynamic events during endocytosis and subsequent intracellular trafficking. The methods are based on enlargement of 1.4 nm Nanogold attached to an endocytosed ligand. Nanogold, a small label that does not induce misdirection of ligand-receptor complexes, is ideal for labeling ligands endocytosed by live cells, but is too small to be routinely located in cells by electron microscopy. Traditional pre-embedding enhancement protocols to enlarge Nanogold are not compatible with high pressure freezing/freeze substitution fixation (HPF/FSF), the most accurate method to preserve ultrastructure and dynamic events during trafficking. We have developed an improved enhancement procedure for chemically fixed samples that reduced auto-nucleation, and a new pre-embedding gold enlarging technique for HPF/FSF samples that preserved contrast and ultrastructure and can be used for high-resolution tomography. We evaluated our methods using labeled Fc as a ligand for the neonatal Fc receptor. Attachment of Nanogold to Fc did not interfere with receptor binding or uptake, and gold-labeled Fc could be specifically enlarged to allow identification in 2D projections and in tomograms. These methods should be broadly applicable to many endocytosis and transcytosis studies.
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Endocitosis , Congelación , Oro/química , Nanopartículas del Metal , Microscopía Electrónica/métodos , Animales , Animales Recién Nacidos , Concentración de Iones de Hidrógeno , Ratas , Ratas Sprague-DawleyRESUMEN
The neonatal Fc receptor (FcRn) transports IgG across epithelial cell barriers to provide maternal antibodies to offspring and serves as a protection receptor by rescuing endocytosed IgG and albumin from lysosomal degradation. Here we describe the generation of polarized Madin-Darby canine kidney (MDCK) cells expressing rat FcRn (rFcRn) to investigate the potential requirement for ligand bivalency in FcRn-mediated transport. The rFcRn-MDCK cells bind, internalize and bidirectionally transcytose the bivalent ligands IgG and Fc across polarized cell monolayers. However, they cannot be used to study FcRn-mediated transport of the monovalent ligand albumin, as we observe no specific binding, internalization or transcytosis of rat albumin. To address whether ligand bivalency is required for transport, the ability of rFcRn to transcytose and recycle wild-type Fc homodimers (wtFc; two FcRn-binding sites) and a heterodimeric Fc (hdFc; one FcRn-binding site) was compared. We show that ligand bivalency is not required for transcytosis or recycling, but that wtFc is transported more efficiently than hdFc, particularly at lower concentrations. We also demonstrate that hdFc and wtFc have different intracellular fates, with more hdFc than wtFc being trafficked to lysosomes and degraded, suggesting a role for avidity effects in FcRn-mediated IgG transport.