RESUMEN
A transcellular shuttle system was generated for the delivery of non-covalently linked payloads across blood-brain barrier (BBB) endothelial cells. Transcytosis-enabling shuttles are composed of bispecific antibodies (bsAbs) that simultaneously bind transferrin receptor (TfR) and haptens such as digoxigenin or biocytinamide. Haptenylated payloads are attached to these vehicles via non-covalent hapten-antibody complexation. This enables targeting to and internalization into human BBB-derived microvascular endothelial hCMEC/D3 cells. In contrast to other shuttles, this system does not require special affinities or formats of their TfR-binding moieties for transcytosis and subsequent release. Non-covalent payload complexation to bsAb is flexible and robust, works for a multitude of payloads and enables separation of payloads from shuttles during transcytosis. Released payloads can enter the brain without connected bsAb entities, minimizing potential interference with distribution or functionality. Intracellular separation of shuttle and payload and recycling to cell surfaces may also enable recharging of the cell-bound BBB shuttle with payload for subsequent (merry-go-round) transport cycles.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Transcitosis , Línea Celular , Células HEK293 , HumanosRESUMEN
Humanized hapten-binding IgGs were designed with an accessible cysteine close to their binding pockets, for specific covalent payload attachment. Individual analyses of known structures of digoxigenin (Dig)- and fluorescein (Fluo) binding antibodies and a new structure of a biotin (Biot)-binder, revealed a "universal" coupling position (52(+2)) in proximity to binding pockets but without contributing to hapten interactions. Payloads that carry a free thiol are positioned on the antibody and covalently linked to it via disulfides. Covalent coupling is achieved and driven toward complete (95-100%) payload occupancy by spontaneous redox shuffling between antibody and payload. Attachment at the universal position works with different haptens, antibodies, and payloads. Examples are the haptens Fluo, Dig, and Biot combined with various fluorescent or peptidic payloads. Disulfide-bonded covalent antibody-payload complexes do not dissociate in vitro and in vivo. Coupling requires the designed cysteine and matching payload thiol because payload or antibody without the Cys/thiol are not linked (<5% nonspecific coupling). Hapten-mediated positioning is necessary as hapten-thiol-payload is only coupled to antibodies that bind matching haptens. Covalent complexes are more stable in vivo than noncovalent counterparts because digoxigeninylated or biotinylated fluorescent payloads without disulfide-linkage are cleared more rapidly in mice (approximately 50% reduced 48 hour serum levels) compared with their covalently linked counterparts. The coupling technology is applicable to many haptens and hapten binding antibodies (confirmed by automated analyses of the structures of 140 additional hapten binding antibodies) and can be applied to modulate the pharmacokinetics of small compounds or peptides. It is also suitable to link payloads in a reduction-releasable manner to tumor- or tissue-targeting delivery vehicles.
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Anticuerpos/inmunología , Disulfuros/inmunología , Haptenos/inmunología , Fragmentos de Péptidos/inmunología , Animales , Anticuerpos/química , Anticuerpos/metabolismo , Disulfuros/química , Disulfuros/metabolismo , Haptenos/química , Haptenos/metabolismo , Ratones , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Compuestos de Sulfhidrilo/química , Compuestos de Sulfhidrilo/inmunología , Compuestos de Sulfhidrilo/metabolismoRESUMEN
Bispecific antibodies that bind cell-surface targets as well as digoxigenin (Dig) were generated for targeted payload delivery. Targeting moieties are IgGs that bind the tumor antigens Her2, IGF1R, CD22, or LeY. A Dig-binding single-chain Fv was attached in disulfide-stabilized form to C termini of CH3 domains of targeting antibodies. Bispecific molecules were expressed in mammalian cells and purified in the same manner as unmodified IgGs. They are stable without aggregation propensity and retain binding specificity/affinity to cell-surface antigens and Dig. Digoxigeninylated payloads were generated that retain full functionality and can be complexed to bispecific antibodies in a defined 21 ratio. Payloads include small compounds (Dig-Cy5, Dig-Doxorubicin) and proteins (Dig-GFP). Complexed payloads are targeted by the bispecifics to cancer cells and because these complexes are stable in serum, they can be applied for targeted delivery. Because Dig bispecifics also effectively capture digoxigeninylated compounds under physiological conditions, separate administration of uncharged Dig bispecifics followed by application of Dig payload is sufficient to achieve antibody-mediated targeting in vitro and in vivo.
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Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/administración & dosificación , Digoxigenina/inmunología , Sistemas de Liberación de Medicamentos/métodos , Anticuerpos Biespecíficos/inmunología , Antígenos de Neoplasias/inmunología , Carbocianinas/administración & dosificación , Línea Celular Tumoral , Doxorrubicina/administración & dosificación , Proteínas Fluorescentes Verdes/administración & dosificación , Humanos , Métodos , Anticuerpos de Cadena ÚnicaRESUMEN
In this study we present novel bispecific antibodies that simultaneously target the insulin-like growth factor receptor type I (IGF-1R) and epidermal growth factor receptor (EGFR). For this purpose disulfide stabilized scFv domains of the EGFR/ADCC antibody GA201 were fused via serine-glycine connectors to the C-terminus of the heavy (XGFR2) or light chain (XGFR4), or the N-termini of the light (XGFR5) or heavy chain (XGFR3) of the IGF-1R antibody R1507 as parental IgG1 antibody. The resulting bispecific IGF-1R-EGFR antibodies XGFR2, XGFR3 and XGFR4 were successfully generated with yields and stability comparable to conventional IgG1 antibodies. They effectively inhibited IGF-1R and EGFR phosphorylation and 3D proliferation of H322M and H460M2 tumor cells, induced strong down-modulation of IGF-1R as well as enhanced EGFR down-modulation compared to the parental EGFR antibody GA201 and were ADCC competent. The bispecific XGFR derivatives showed a strong format dependent influence of N- or C-terminal heavy and light chain scFv attachment on ADCC activity and an increase in receptor downregulation over the parental combination in vitro. XGFR2 and XGFR4 were selected for in vivo evaluation and showed potent anti-tumoral efficacy comparable to the combination of monospecific IGF-1R and EGFR antibodies in subcutaneous BxPC3 and H322M xenograft models. In summary, we have managed to overcome issues of stability and productivity of bispecific antibodies, discovered important antibody fusion protein design related differences on ADCC activity and receptor downmodulation and show that IGF-1R-EGFR antibodies represent an attractive therapeutic strategy to simultaneously target two key components de-regulated in multiple cancer types, with the ultimate goal to avoid the formation of resistance to therapy.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos Biespecíficos/uso terapéutico , Receptores ErbB/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/uso terapéutico , Neoplasias/terapia , Receptor IGF Tipo 1/inmunología , Animales , Anticuerpos Biespecíficos/genética , Afinidad de Anticuerpos , Línea Celular , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Clonación Molecular , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunoglobulina G/genética , Inmunoterapia , Ratones , Ratones SCID , Modelos Moleculares , Neoplasias/inmunología , Neoplasias/metabolismo , Fosforilación/efectos de los fármacos , Ingeniería de Proteínas , Receptor IGF Tipo 1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/uso terapéutico , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/uso terapéuticoRESUMEN
OBJECTIVES: The recently introduced ultrasonic osteotome procedure is an alternative to conventional methods of osteotomy. The aim of the present study was to establish the differences between five recently introduced ultrasonic osteotomes and to perform micromorphological and quantitative roughness analyses of osteotomized bone surfaces. MATERIALS AND METHODS: Fresh, standard-sized bony samples were taken from a rabbit skull using the following ultrasonic osteotomes: the Piezosurgery 3 with insert tip OT7, Piezosurgery Medical with insert tip MT1-10, Piezon Master Surgery with insert tip SL1, VarioSurg with inert tip SG1, and Piezotome 2 with insert tip BS1 II. The required duration of time for each osteotomy was recorded. The prepared surfaces were examined via light microscopy, environmental surface electron microscopy (ESEM), and confocal laser scanning microscopy (CLSM). RESULTS: All of the investigated piezoelectric osteotomes preserved the anatomical structure of bone. The mean roughness values of the osteotomized bone edge obtained using the investigated piezoelectric osteotomes were as follows: 2.47 µm (Piezosurgery 3), 9.79 µm (Piezosurgery Medical), 4.66 µm (Piezon Master Surgery), 6.38 µm (VarioSurg), and 6.06 µm (Piezotome 2). Significantly higher roughness values were observed when using the Piezosurgery Medical in comparison with those achieved by the Piezosurgery 3 (P<0.0001) and Piezon Master Surgery (P=0.002). Different osteotomy durations were achieved using the different piezoelectric osteotomes: 144 s (Piezosurgery 3), 126 s (Piezosurgery Medical), 142 s (Piezon Master Surgery), 149 s (VarioSurg), and 137 s (Piezotome 2). CONCLUSIONS: In the present study, micromorphological differences following the use of various ultrasonic devices were clearly identified. According to this study, it can be concluded that the power and the composition of the teeth of the insert tip might impact procedure duration and cutting qualities.
Asunto(s)
Craneotomía/instrumentación , Ultrasonido , Animales , Femenino , Microscopía Confocal , Microscopía Electrónica de Rastreo , Conejos , Estadísticas no Paramétricas , Propiedades de SuperficieRESUMEN
Obtaining a dynamic population distribution is key to many decision-making processes such as urban planning, disaster management and most importantly helping the government to better allocate socio-technical supply. For the aspiration of these objectives, good population data is essential. The traditional method of collecting population data through the census is expensive and tedious. In recent years, statistical and machine learning methods have been developed to estimate population distribution. Most of the methods use data sets that are either developed on a small scale or not publicly available yet. Thus, the development and evaluation of new methods become challenging. We fill this gap by providing a comprehensive data set for population estimation in 98 European cities. The data set comprises a digital elevation model, local climate zone, land use proportions, nighttime lights in combination with multi-spectral Sentinel-2 imagery, and data from the Open Street Map initiative. We anticipate that it would be a valuable addition to the research community for the development of sophisticated approaches in the field of population estimation.
RESUMEN
In this study, we describe novel tetravalent, bispecific antibody derivatives that bind two different epitopes on the HIV coreceptor CCR5. The basic protein formats that we applied were derived from Morrison-type bispecific antibodies: whole IgGs to which we connected single-chain antibodies (scFvs) via (Gly4Ser)n sequences at either the C or N terminus of the light chain or heavy chain. By design optimization, including disulfide stabilization of scFvs or introduction of 30-amino-acid linkers, stable molecules could be obtained in amounts that were within the same range as or no less than 4-fold lower than those observed with monoclonal antibodies in transient expression assays. In contrast to monospecific CCR5 antibodies, bispecific antibody derivatives block two alternative docking sites of CCR5-tropic HIV strains on the CCR5 coreceptor. Consequently, these molecules showed 18- to 57-fold increased antiviral activities compared to the parent antibodies. Most importantly, one prototypic tetravalent CCR5 antibody had antiviral activity against virus strains resistant to the single parental antibodies. In summary, physical linkage of two CCR5 antibodies targeting different epitopes on the HIV coreceptor CCR5 resulted in tetravalent, bispecific antibodies with enhanced antiviral potency against wild-type and CCR5 antibody-resistant HIV-1 strains.
Asunto(s)
Anticuerpos Biespecíficos/farmacología , VIH-1/efectos de los fármacos , Receptores CCR5/inmunología , Línea Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , VIH-1/inmunología , HumanosRESUMEN
OBJECTIVES: The purpose of this study was to evaluate the intraosseous temperature changes during ultrasonic and conventional implant site preparation in vitro with respect to the effect of load and irrigation volume. MATERIAL AND METHODS: Implant sites were prepared using two different ultrasonic devices (Piezosurgery, Mectron Medical Technology and VarioSurg, NSK) and one conventional device (Straumann) at loads of 5, 8, 15 and 20 N and with irrigation volumes of 20, 50 and 80 ml/min. During implant site preparation, temperatures were measured in fresh, equally tempered bovine ribs using two thermocouples placed at a distance of 1.5 mm around the drilling site in cortical and cancellous bone. The preparation time was recorded. RESULTS: The heat production and time required for implant site preparation using both ultrasonic devices were significantly higher than those for conventional drilling (P<0.01). Increased loading had no effect on heat production. A higher irrigation volume was associated with a diminished temperature increase in the cortical bone for ultrasonic but not for conventional drilling, which resulted in significantly lower temperatures in cortical as compared with cancellous bone during ultrasonic implant site preparation. CONCLUSIONS: Ultrasonic implant site preparation is more time consuming and generates higher bone temperatures than conventional drilling. However, with the levels of irrigation, ultrasonic implant site preparation can be an equally safe method.
Asunto(s)
Implantación Dental Endoósea , Calor , Osteotomía/instrumentación , Piezocirugía/instrumentación , Animales , Bovinos , Técnica Odontológica de Alta Velocidad , Humanos , Costillas , Estadísticas no ParamétricasRESUMEN
Simulating a viral infection in tumor cells is an attractive concept to eliminate tumor cells. We previously reported the molecular design and the in vitro potency of recombinant monoclonal antibodies fused to a virus-derived peptide MHC class I complex that bypass the peptide processing and MHC loading pathway and directly displays a viral peptide in an MHC class I complex on the tumor cell surface. Here, we show that a vaccination-induced single peptide-specific CD8 T cell response was sufficient to eliminate B16 melanoma tumor cells in vivo in a fully immunocompetent, syngeneic mouse tumor model when mice were treated with mouse pMHCI-IgGs fusion proteins targeting the mouse fibroblast activation protein. Tumor growth of small, established B16 lung metastases could be controlled. The pMHCI-IgG had similar potency as an analogous pan-CD3 T-cell bispecific antibody. In contrast to growth control of small tumors, none of the compounds controlled larger solid tumors of MC38 cancer cells, despite penetration of pMHCI-IgGs into the tumor tissue and clear attraction and activation of antigen-specific CD8 T cells inside the tumor. pMHCI-IgG can have a similar potency as classical pan-T-cell recruiting molecules. The results also highlight the need to better understand immune suppression in advanced solid tumors.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Vacunas contra el Cáncer/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Inmunoglobulina G/inmunología , Melanoma Experimental/inmunología , Animales , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunologíaRESUMEN
Generation of bispecific antibodies (bsAbs) requires a combination of compatible binders in formats that support desired functionalities. Here, we report that bsAb-matrices can be generated by Format Chain Exchange (FORCE), enabling screening of combinatorial binder/format spaces. Input molecules for generation of bi/multi-valent bsAbs are monospecific entities similar to knob-into-hole half-antibodies, yet with complementary CH3-interface-modulated and affinity-tagged dummy-chains. These contain mutations that lead to limited interface repulsions without compromising expression or biophysical properties of educts. Mild reduction of combinations of educts triggers spontaneous chain-exchange reactions driven by partially flawed CH3-educt interfaces resolving to perfect complementarity. This generates large bsAb matrices harboring different binders in multiple formats. Benign biophysical properties and good expression yields of educts, combined with simplicity of purification enables process automation. Examples that demonstrate the relevance of screening binder/format combinations are provided as a matrix of bsAbs that simultaneously bind Her1/Her2 and DR5 without encountering binder or format-inflicted interferences.
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Anticuerpos Biespecíficos/biosíntesis , Ensayos Analíticos de Alto Rendimiento , Anticuerpos Biespecíficos/aislamiento & purificación , Automatización , Células HEK293 , Humanos , Mutación/genética , Multimerización de ProteínaRESUMEN
Soluble forms of CD83, a dendritic cell-specific surface glycoprotein, have been strongly proposed to be of therapeutic utility in inflammatory conditions such as multiple sclerosis and transplantation. We demonstrate here, however, that eukaryotically expressed, recombinant soluble human CD83-Ig molecules fail to achieve efficacy in model systems for those conditions: mouse experimental autoimmune encephalomyelitis models in vivo or in mixed lymphocyte reactions in vitro. These results raise concern as to the viability of a eukaryotically expressed soluble CD83 strategy for clinical therapeutic use.
Asunto(s)
Antígenos CD/inmunología , Antígenos CD/uso terapéutico , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inmunoglobulinas/inmunología , Inmunoglobulinas/uso terapéutico , Prueba de Cultivo Mixto de Linfocitos , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/uso terapéutico , Animales , Antígenos CD/sangre , Células Dendríticas/inmunología , Encefalomielitis Autoinmune Experimental/inmunología , Encefalomielitis Autoinmune Experimental/metabolismo , Humanos , Inmunoglobulinas/sangre , Glicoproteínas de Membrana/sangre , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/uso terapéutico , Antígeno CD83RESUMEN
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
Bifunctional antibody fusion proteins engaging effector T cells for targeted elimination of tumor cells via CD3 binding have shown efficacy in both preclinical and clinical studies. Different from such a polyclonal T-cell recruitment, an alternative concept is to engage only antigen-specific T-cell subsets. Recruitment of specific subsets of T cells may be as potent but potentially lead to fewer side effects. Tumor-targeted peptide-MHC class I complexes (pMHCI-IgGs) bearing known antigenic peptides complexed with MHC class I molecules mark tumor cells as antigenic and utilize the physiologic way to interact with and activate T-cell receptors. If, for example, virus-specific CD8(+) T cells are addressed, the associated strong antigenicity and tight immune surveillance of the effector cells could lead to efficacious antitumor treatment in various tissues. However, peptide-MHC class I fusions are difficult to express recombinantly, especially when fused to entire antibody molecules. Consequently, current formats are largely limited to small antibody fragment fusions expressed in bacteria followed by refolding or chemical conjugation. Here, we describe a new molecular format bearing a single pMHCI complex per IgG fusion molecule characterized by enhanced stability and expression yields. This molecular format can be expressed in a full immunoglobulin format and can be designed as mono- or bivalent antibody binders. Mol Cancer Ther; 15(9); 2130-42. ©2016 AACR.
Asunto(s)
Anticuerpos Biespecíficos/inmunología , Anticuerpos/inmunología , Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Animales , Línea Celular Tumoral , Citotoxicidad Inmunológica , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Antígenos de Histocompatibilidad Clase I/metabolismo , Humanos , Inmunoglobulina G/inmunología , Activación de Linfocitos/inmunología , Ratones , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/patología , Péptidos/inmunología , Péptidos/metabolismo , Fosfoproteínas/química , Fosfoproteínas/inmunología , Fosfoproteínas/metabolismo , Unión Proteica , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Proteínas de la Matriz Viral/química , Proteínas de la Matriz Viral/inmunología , Proteínas de la Matriz Viral/metabolismoRESUMEN
Tumor cells escape immune eradication through multiple mechanisms, including loss of antigenicity and local suppression of effector lymphocytes. To counteract these obstacles, we aimed to direct the unique cytomegalovirus (CMV)-specific immune surveillance against tumor cells. We developed a novel generation of fusion proteins composed of a tumor antigen-specific full immunoglobulin connected to a single major histocompatibility class I complex bearing a covalently linked virus-derived peptide (pMHCI-IgG). Here, we show that tumor antigen-expressing cancer cells, which are decorated with pMHCI-IgGs containing a HLA-A*0201 molecule associated with a CMV-derived peptide, are specifically eliminated through engagement of antigen-specific CD8(+) T cells isolated from peripheral blood mononuclear cell preparations of CMV-infected humans. These CD8(+) T cells act without additional expansion, preactivation, or provision of costimulatory signals. Elimination of tumor cells is induced at similar concentrations and with similar time kinetics as those seen with bispecific T-cell engagers (BiTE). However, while BiTE-like reagents indiscriminately activate T cells through binding to the T-cell receptor complex, pMHCI-IgGs selectively engage antigen-specific, constantly renewable, differentiated effector cytotoxic T lymphocytes to tumor cells, thereby representing a novel class of anticancer immunotherapeutics with potentially improved safety and efficacy profiles.
Asunto(s)
Citomegalovirus/inmunología , Antígeno HLA-A2/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Leucocitos Mononucleares/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Anticuerpos/inmunología , Línea Celular Tumoral , Humanos , Activación de Linfocitos/inmunología , Ratones , Péptidos/inmunologíaRESUMEN
The N-terminal, extracellular domain of the receptor for glucagon-like peptide 1 (GLP-1 receptor) was expressed at a high level in E. coli and isolated as inclusion bodies. Renaturation with concomitant disulfide bond formation was achieved from guanidinium-solubilized material. A soluble and active fraction of the protein was isolated by ion exchange chromatography and gel filtration. Complex formation with GLP-1 was shown by cross-linking experiments, surface plasmon resonance measurements, and isothermal titration calorimetry. The existence of disulfide bridges in the N-terminal receptor fragment was proven after digestion of the protein with pepsin. Further analysis revealed a disulfide-binding pattern with links between cysteines 46 and 71, 62 and 104, and between 85 and 126.
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Disulfuros/química , Pliegue de Proteína , Receptores de Glucagón/química , Secuencia de Aminoácidos , Calorimetría , Dicroismo Circular , Escherichia coli/genética , Escherichia coli/metabolismo , Glucagón/metabolismo , Péptido 1 Similar al Glucagón , Receptor del Péptido 1 Similar al Glucagón , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Precursores de Proteínas/metabolismo , Estructura Terciaria de Proteína , Receptores de Glucagón/aislamiento & purificación , Receptores de Glucagón/metabolismo , VolumetríaRESUMEN
Styloid-carotid artery syndrome was first described by Eagle and is associated with cervical and facial pain caused by head movement resulting from mechanical compression of the carotid nerve plexus due to an elongated styloid process. The case of a 49-year-old man with persistent cervical pain, neurological symptoms and an elongated styloid process of 7.5 cm is reported here; this patient was successfully treated using Piezosurgery. In addition, a literature review is included.
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Osificación Heterotópica/cirugía , Piezocirugía/métodos , Arteria Carótida Interna/patología , Nervio Facial/patología , Dolor Facial/cirugía , Estudios de Seguimiento , Humanos , Hueso Hioides/diagnóstico por imagen , Imagenología Tridimensional/métodos , Ligamentos/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Dolor de Cuello/cirugía , Osificación Heterotópica/diagnóstico por imagen , Radiografía Panorámica , Hueso Temporal/anomalías , Hueso Temporal/diagnóstico por imagen , Hueso Temporal/cirugía , Tomografía Computarizada por Rayos X/métodosRESUMEN
We applied noncovalent complexes of digoxigenin (Dig) binding antibodies with digoxigeninylated peptide derivatives to modulate their pharmacokinetic properties. A peptide derivative which activates the Y2R receptor was selectively mono-digoxigeninylated by reacting a NHS-Dig derivative with an ε-amino group of lysine 2. This position tolerates modifications without destroying receptor binding and functionality of the peptide. Dig-peptide derivatives can be loaded onto Dig-binding IgGs in a simple and robust reaction, thereby generating peptide-IgG complexes in a defined two to one molar ratio. This indicates that each antibody arm becomes occupied by one haptenylated peptide. In vitro receptor binding and signaling assays showed that Dig-peptides as well as the peptide-antibody complexes retain better potency than the corresponding pegylated peptides. In vivo analyses revealed prolonged serum half-life of antibody-complexed peptides compared to unmodified peptides. Thus, complexes are of sufficient stability for PK modulation. We observed more prolonged weight reduction in a murine diet-induced obesity (DIO) model with antibody-complexed peptides compared to unmodified peptides. We conclude that antibody-hapten complexation can be applied to modulate the PK of haptenylated peptides and in consequence improve the therapeutic efficacy of therapeutic peptides.
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Digoxigenina/química , Haptenos/química , Inmunoglobulina G/química , Péptidos/química , Animales , Dieta Alta en Grasa , Digoxigenina/sangre , Digoxigenina/farmacocinética , Ingestión de Alimentos/efectos de los fármacos , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/tratamiento farmacológico , Péptidos/farmacocinética , Agonistas del Receptor Purinérgico P2Y/administración & dosificación , Receptores Purinérgicos P2Y/metabolismoRESUMEN
We have designed bispecific antibodies that bind one target (anti-Her3) in a bivalent IgG-like manner and contain one additional binding entity (anti-cMet) composed of one V(H) and one V(L) domain connected by a disulfide bond. The molecules are assembled by fusing a V(H,Cys44) domain via flexible connector peptides to the C-terminus of one H-chain (heavy chain), and a V(L,Cys100) to another H-chain. To ensure heterodimerization during expression in mammalian cells, we introduced complementary knobs-into-holes mutations into the different H-chains. The IgG-shaped trivalent molecules carry as third binding entity one disulfide-stabilized Fv (dsFv) without a linker between V(H) and V(L). Tethering the V(H) and V(L) domains at the C-terminus of the C(H)3 domain decreases the on-rates of the dsFv to target antigens without affecting off-rates. Steric hindrance resolves upon removal of one side of the double connection by proteolysis: this improves flexibility and accessibility of the dsFv and fully restores antigen access and affinity. This technology has multiple applications: (i) in cases where single-chain linkers are not desired, dsFvs without linkers can be generated by addition of furin site(s) in the connector that are processed during expression within mammalian cells; (ii) highly active (toxic) entities which affect expression can be produced as inactive dsFvs and subsequently be activated (e.g. via PreScission cleavage) during purification; (iii) entities can be generated which are targeted by the unrestricted binding entity and can be activated by proteases in target tissues. For example, Her3-binding molecules containing linkers with recognition sequences for matrix metalloproteases or urokinase, whose inactivated cMet binding site is activated by proteolytic processing.
Asunto(s)
Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/metabolismo , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Anticuerpos Biespecíficos/genética , Anticuerpos Biespecíficos/inmunología , Sitios de Unión de Anticuerpos , Línea Celular , Disulfuros/química , Expresión Génica , Humanos , Metaloproteinasa 2 de la Matriz/inmunología , Metaloproteinasa 9 de la Matriz/inmunología , Péptido Hidrolasas/metabolismo , Ingeniería de Proteínas , Proteolisis , Receptor ErbB-3/inmunología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Activador de Plasminógeno de Tipo Uroquinasa/inmunologíaRESUMEN
Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3'end was bound in a 2:1 ratio to the bsAbs. These bsAb-siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC(50) siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.Molecular Therapy - Nucleic Acids (2012) 1, e45; doi:10.1038/mtna.2012.39; published online 18 September 2012.
RESUMEN
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