RESUMEN
A solid understanding of physiology is beneficial in optimizing drug delivery and in the development of clinically predictive models of drug disposition kinetics. Although an abundance of data exists in the literature, it is often confounded by the use of various experimental methods and a lack of consensus in values from different sources. To help address this deficiency, we sought to directly compare three important vascular parameters at the tissue level using the same experimental approach in both mice and rats. Interstitial volume, vascular volume, and blood flow were radiometrically measured in selected harvested tissues of both species by extracellular marker infusion, red blood cell labeling, and rubidium chloride bolus distribution, respectively. The latter two parameters were further compared by whole-body autoradiographic imaging. An overall good interspecies agreement was observed for interstitial volume and blood flow on a weight-normalized basis in most tissues. In contrast, the measured vascular volumes of most rat tissues were higher than for mouse. Mice and rats, the two most commonly utilized rodent species in translational drug development, should not be considered as interchangeable in terms of vascular volume per gram of tissue. This will be particularly critical in biodistribution studies of drugs, as the amount of drug in the residual blood of tissues is often not negligible, especially for biologic drugs (e.g., antibodies) having long circulation half-lives. Physiologically based models of drug pharmacokinetics and/or pharmacodynamics also rely on accurate knowledge of biological parameters in tissues. For tissue parameters with poor interspecies agreement, the significance and possible drivers are discussed.
Asunto(s)
Volumen Sanguíneo/fisiología , Ratones/fisiología , Ratas/fisiología , Animales , Peso Corporal/fisiología , Femenino , Modelos Teóricos , Ratas Sprague-DawleyRESUMEN
The development of clinically relevant preclinical models that mimic the hallmarks of neurodegenerative disease is an ongoing pursuit in early drug development. In particular, robust physiological characterization of central nervous system (CNS) disease models is necessary to predict drug delivery to target tissues and to correctly interpret pharmacodynamic responses to disease-modifying therapeutic candidates. Efficient drug delivery across the blood-CNS barrier is a particularly daunting task, prompting our strategy to evaluate the biodistribution of five distinct molecular probes in a well-characterized mouse model of neurodegeneration. A transgenic mouse model of amyotrophic lateral sclerosis was selected based on a phenotype resembling clinical symptoms, including loss of motor neurons from the spinal cord and paralysis in one or more limbs, due to expression of a G93A mutant form of human superoxide dismutase (SOD1). The tissue distributions of two proteins, albumin and a representative immunoglobulin G antibody, as well as two blood flow markers, the lipophilic blood flow marker Ceretec (i.e., (99m)Tc-HMPAO) and the polar ionic tracer, rubidium-86 chloride ((86)RbCl), were measured following intravenous injection in SOD1(G93A) and age-matched control mice. The radiopharmaceutical TechneScan PYP was also used to measure the distribution of (99m)Tc-labeled red blood cells as a blood pool marker. Both the antibody and (86)Rb were able to cross the blood-spinal cord barrier in SOD1(G93A) mice to a greater extent than in control mice. Although the biodistribution patterns of antibody, albumin, and RBCs were largely similar, notable differences were detected in muscle and skin. Moreover, vastly different biodistribution patterns were observed for a lipophilic and polar perfusion agent, with SOD1(G93A) mutation resulting in reduced renal filtration rates for the former but not the latter. Overall, the multiprobe strategy provided an opportunity to efficiently collect an abundance of physiological information, including the degree and regional extent of blood-CNS barrier permeability, in a preclinical model of neurodegeneration.
Asunto(s)
Degeneración Nerviosa/fisiopatología , Esclerosis Amiotrófica Lateral/diagnóstico por imagen , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Volumen Sanguíneo , Barrera Hematoencefálica/fisiología , Circulación Cerebrovascular , Cloruros/farmacocinética , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Femenino , Humanos , Inmunoglobulina G/metabolismo , Ratones , Ratones Mutantes , Ratones Transgénicos , Transporte de Proteínas , Cintigrafía , Radiofármacos/farmacocinética , Rubidio/farmacocinética , Radioisótopos de Rubidio/farmacocinética , Superóxido Dismutasa/genética , Exametazima de Tecnecio Tc 99m/farmacocinética , Distribución TisularRESUMEN
Antibody-drug conjugates (ADCs) are designed to combine the exquisite specificity of antibodies to target tumor antigens with the cytotoxic potency of chemotherapeutic drugs. In addition to the general chemical stability of the linker, a thorough understanding of the relationship between ADC composition and biological disposition is necessary to ensure that the therapeutic window is not compromised by altered pharmacokinetics (PK), tissue distribution, and/or potential organ toxicity. The six-transmembrane epithelial antigen of prostate 1 (STEAP1) is being pursued as a tumor antigen target. To assess the role of ADC composition in PK, we evaluated plasma and tissue PK profiles in rats, following a single dose, of a humanized anti-STEAP1 IgG1 antibody, a thio-anti-STEAP1 (ThioMab) variant, and two corresponding thioether-linked monomethylauristatin E (MMAE) drug conjugates modified through interchain disulfide cysteine residues (ADC) and engineered cysteines (TDC), respectively. Plasma PK of total antibody measured by enzyme-linked immunosorbent assay (ELISA) revealed â¼45% faster clearance for the ADC relative to the parent antibody, but no apparent difference in clearance between the TDC and unconjugated parent ThioMab. Total antibody clearances of the two unconjugated antibodies were similar, suggesting minimal effects on PK from cysteine mutation. An ELISA specific for MMAE-conjugated antibody indicated that the ADC cleared more rapidly than the TDC, but total antibody ELISA showed comparable clearance for the two drug conjugates. Furthermore, consistent with relative drug load, the ADC had a greater magnitude of drug deconjugation than the TDC in terms of free plasma MMAE levels. Antibody conjugation had a noticeable, albeit minor, impact on tissue distribution with a general trend toward increased hepatic uptake and reduced levels in other highly vascularized organs. Liver uptakes of ADC and TDC at 5 days postinjection were 2-fold and 1.3-fold higher, respectively, relative to the unmodified antibodies. Taken together, these results indicate that the degree of overall structural modification in anti-STEAP1-MMAE conjugates has a corresponding level of impact on both PK and tissue distribution.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Antígenos de Neoplasias/inmunología , Inmunoconjugados/química , Inmunoconjugados/farmacocinética , Oligopéptidos/química , Oligopéptidos/farmacocinética , Oxidorreductasas/inmunología , Animales , Anticuerpos Monoclonales/sangre , Disulfuros/química , Humanos , Inmunoconjugados/sangre , Masculino , Modelos Moleculares , Oligopéptidos/sangre , Ratas , Ratas Sprague-Dawley , Distribución TisularRESUMEN
The DNA-binding alpha/beta-type small acid-soluble proteins (SASPs) are a major factor in the resistance and long-term survival of spores of Bacillus species by protecting spore DNA against damage due to desiccation, heat, toxic chemicals, enzymes, and UV radiation. We now report the crystal structure at 2.1 A resolution of an alpha/beta-type SASP bound to a 10-bp DNA duplex. In the complex, the alpha/beta-type SASP adopt a helix-turn-helix motif, interact with DNA through minor groove contacts, bind to approximately 6 bp of DNA as a dimer, and the DNA is in an A-B type conformation. The structure of the complex provides important insights into the molecular details of both DNA and alpha/beta-type SASP protection in the complex and thus also in spores.
Asunto(s)
Bacillus/genética , Proteínas Bacterianas/metabolismo , Daño del ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Esporas Bacterianas/genética , Proteínas Bacterianas/genética , Cristalización , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Conformación de Ácido Nucleico , Conformación ProteicaRESUMEN
Streptococcus pneumoniae open reading frame SP1492 encodes a surface protein that contains a novel conserved domain similar to the repeated fragments of mucin-binding proteins from lactobacilli and lactococci. To investigate the functional role(s) of this protein and its potential adhesive properties, the surface-exposed region of SP1492 was expressed in Escherichia coli, purified to homogeneity, and partially characterized by biophysical and immunological methods. Circular dichroism and sedimentation measurements confirmed that SP1492 is an all-beta protein that exists in solution as a monomer. The SP1492 protein has been shown to be expressed by S. pneumoniae and was experimentally localized to its surface. The protein functional domain binds to mucins II and III from porcine stomach and to purified submaxillary bovine gland mucin. It appears to be one of the very few unambiguous pneumococcal adhesin molecules known to date. A hypothetical model constructed by ab initio techniques predicts a novel beta-sandwich protein structure.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Genoma Bacteriano , Mucinas/metabolismo , Streptococcus pneumoniae/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Cromatografía en Gel , Dicroismo Circular , Clonación Molecular , Biología Computacional , Regulación Bacteriana de la Expresión Génica , Ingeniería Genética , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Análisis de Secuencia de ProteínaRESUMEN
An engineered variant of an alpha/beta-type small acid-soluble spore protein (SASP) from Bacillus subtilis was crystallized in a complex with a ten-base-pair double-stranded DNA by the hanging-drop vapor-diffusion method using ammonium sulfate as a precipitating agent. Crystals grew at 281 K using sodium cacodylate buffer pH 5.5 and these crystals diffracted X-rays to beyond 2.4 A resolution using synchrotron radiation. The crystallized complex contains two or three SASP molecules bound to one DNA molecule. The crystals belong to the hexagonal space group P6(1)22 or P6(5)22, with unit-cell parameters a = b = 87.0, c = 145.4 A, alpha = beta = 90.0, gamma = 120.0 degrees. Diffraction data were 96.6% complete to 2.4 A resolution, with an R(sym) of 8.5%. Structure solution by the multiwavelength/single-wavelength anomalous dispersion method using isomorphous crystals of selenomethionine-labeled protein is in progress.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , ADN Bacteriano/química , Bacillus subtilis/genética , Proteínas Bacterianas/genética , Cristalización , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Esporas Bacterianas/química , Esporas Bacterianas/genética , Difracción de Rayos XRESUMEN
MPDL3280A is a human monoclonal antibody that targets programmed cell death-1 ligand 1 (PD-L1), and exerts anti-tumor activity mainly by blocking PD-L1 interaction with programmed cell death-1 (PD-1) and B7.1. It is being investigated as a potential therapy for locally advanced or metastatic malignancies. The purpose of the study reported here was to characterize the pharmacokinetics, pharmacodynamics, tissue distribution and tumor penetration of MPDL3280A and/or a chimeric anti-PD-L1 antibody PRO304397 to help further clinical development. The pharmacokinetics of MPDL3280A in monkeys at 0.5, 5 and 20 mg · kg(-1) and the pharmacokinetics / pharmacodynamics of PRO304397 in mice at 1, 3 10 mg · kg(-1) were determined after a single intravenous dose. Tissue distribution and tumor penetration for radiolabeled PRO304397 in tumor-bearing mouse models were determined. The pharmacokinetics of MPDL3280A and PRO304397 were nonlinear in monkeys and mice, respectively. Complete saturation of PD-L1 in blood in mice was achieved at serum concentrations of PRO304397 above â¼ 0.5 µg · mL(-1). Tissue distribution and tumor penetration studies of PRO304397 in tumor-bearing mice indicated that the minimum tumor interstitial to plasma radioactivity ratio was â¼ 0.3; saturation of target-mediated uptake in non-tumor tissues and desirable exposure in tumors were achieved at higher serum concentrations, and the distribution into tumors was dose-and time-dependent. The biodistribution data indicated that the efficacious dose is mostly likely higher than that estimated based on simple pharmacokinetics/pharmacodynamics in blood. These data also allowed for estimation of the target clinical dose for further development of MPDL3280A.
Asunto(s)
Anticuerpos Monoclonales , Anticuerpos Antineoplásicos , Antígeno B7-H1/antagonistas & inhibidores , Puntos de Control del Ciclo Celular , Proteínas de Neoplasias/antagonistas & inhibidores , Neoplasias Experimentales , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales/farmacología , Anticuerpos Antineoplásicos/inmunología , Anticuerpos Antineoplásicos/farmacología , Antígeno B7-H1/inmunología , Células CHO , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/inmunología , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Femenino , Humanos , Macaca fascicularis , Ratones , Ratones Endogámicos BALB C , Metástasis de la Neoplasia , Proteínas de Neoplasias/inmunología , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/patologíaRESUMEN
The blood-brain barrier (BBB) poses a major challenge for developing effective antibody therapies for neurological diseases. Using transcriptomic and proteomic profiling, we searched for proteins in mouse brain endothelial cells (BECs) that could potentially be exploited to transport antibodies across the BBB. Due to their limited protein abundance, neither antibodies against literature-identified targets nor BBB-enriched proteins identified by microarray facilitated significant antibody brain uptake. Using proteomic analysis of isolated mouse BECs, we identified multiple highly expressed proteins, including basigin, Glut1, and CD98hc. Antibodies to each of these targets were significantly enriched in the brain after administration in vivo. In particular, antibodies against CD98hc showed robust accumulation in brain after systemic dosing, and a significant pharmacodynamic response as measured by brain Aß reduction. The discovery of CD98hc as a robust receptor-mediated transcytosis pathway for antibody delivery to the brain expands the current approaches available for enhancing brain uptake of therapeutic antibodies.
Asunto(s)
Anticuerpos/uso terapéutico , Transporte Biológico/fisiología , Barrera Hematoencefálica/metabolismo , Encéfalo/metabolismo , Receptores de Transferrina/metabolismo , Animales , Anticuerpos/inmunología , Células Endoteliales/metabolismo , Cadena Pesada de la Proteína-1 Reguladora de Fusión/inmunología , Ratones , Proteómica/métodos , Transcitosis/fisiologíaRESUMEN
The spread of tau pathology correlates with cognitive decline in Alzheimer's disease. In vitro, tau antibodies can block cell-to-cell tau spreading. Although mechanisms of anti-tau function in vivo are unknown, effector function might promote microglia-mediated clearance. In this study, we investigated whether antibody effector function is required for targeting tau. We compared efficacy in vivo and in vitro of two versions of the same tau antibody, with and without effector function, measuring tau pathology, neuron health, and microglial function. Both antibodies reduced accumulation of tau pathology in Tau-P301L transgenic mice and protected cultured neurons against extracellular tau-induced toxicity. Only the full-effector antibody enhanced tau uptake in cultured microglia, which promoted release of proinflammatory cytokines. In neuron-microglia co-cultures, only effectorless anti-tau protected neurons, suggesting full-effector tau antibodies can induce indirect toxicity via microglia. We conclude that effector function is not required for efficacy, and effectorless tau antibodies may represent a safer approach to targeting tau.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Microglía/metabolismo , Proteínas tau/metabolismo , Enfermedad de Alzheimer/patología , Animales , Anticuerpos/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Células Cultivadas , Técnicas de Cocultivo/métodos , Citocinas/metabolismo , Ratones Transgénicos , Neuronas/metabolismoRESUMEN
DM1, a derivative of maytansine, is the cytotoxic component of the antibody-drug conjugate trastuzumab emtansine (T-DM1). Understanding the disposition and metabolism of DM1 would help to assess (1) any tissue-specific distribution and risk for potential drug-drug interactions and (2) the need for special patient population studies. To this end, the current study determined the disposition and metabolism of DM1 following single intravenous administration of [(3)H]-DM1 in Sprague Dawley rats. Blood, tissues, urine, bile, and feces were collected up to 5 days after dose administration and analyzed for total radioactivity and metabolites. Results showed that radioactivity cleared rapidly from the blood and quickly distributed to the lungs, liver, kidneys, spleen, heart, gastrointestinal tract, adrenal glands, and other tissues without significant accumulation or persistence. The majority of dosed radioactivity was recovered in feces (~100% of the injected dose over 5 days) with biliary elimination being the predominant route (~46% of the injected dose over 3 days). Excretion in urine was minimal (~5% of the injected dose over 5 days). Mass balance was achieved over 5 days. An analysis of bile samples revealed a small fraction of intact DM1 and a predominance of DM1 metabolites formed through oxidation, hydrolysis, S-methylation, and glutathione and its related conjugates. Collectively, these data demonstrate that DM1 is extensively distributed and quickly cleared from blood, and undergoes extensive metabolism to form multiple metabolites, which are mainly eliminated through the hepatic-biliary route, suggesting that hepatic function (but not renal function) plays an important role in DM1 elimination.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Antineoplásicos/farmacocinética , Hígado/metabolismo , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Anticuerpos Monoclonales Humanizados/sangre , Antineoplásicos/administración & dosificación , Antineoplásicos/sangre , Bilis/metabolismo , Biotransformación , Heces/química , Femenino , Glutatión/metabolismo , Eliminación Hepatobiliar , Hidrólisis , Inyecciones Intravenosas , Maitansina/administración & dosificación , Maitansina/sangre , Maitansina/farmacocinética , Metilación , Estructura Molecular , Oxidación-Reducción , Ratas Sprague-Dawley , Eliminación Renal , Distribución Tisular , TrastuzumabRESUMEN
Subcutaneous (SC) injection is becoming a more common route for the administration of biopharmaceuticals. Currently, there is no reliable in vitro method that can be used to anticipate the in vivo performance of a biopharmaceutical formulation intended for SC injection. Nor is there an animal model that can predict in vivo outcomes such as bioavailability in humans. We address this unmet need by the development of a novel in vitro system, termed Scissor (Subcutaneous Injection Site Simulator). The system models environmental changes that a biopharmaceutical could experience as it transitions from conditions of a drug product formulation to the homeostatic state of the hypodermis following SC injection. Scissor uses a dialysis-based injection chamber, which can incorporate various concentrations and combinations of acellular extracellular matrix (ECM) components that may affect the release of a biopharmaceutical from the SC injection site. This chamber is immersed in a container of a bicarbonate-based physiological buffer that mimics the SC injection site and the infinite sink of the body. Such an arrangement allows for real-time monitoring of the biopharmaceutical within the injection chamber, and can be used to characterize physicochemical changes of the drug and its interactions with ECM components. Movement of a biopharmaceutical from the injection chamber to the infinite sink compartment simulates the drug migration from the injection site and uptake by the blood and/or lymph capillaries. Here, we present an initial evaluation of the Scissor system using the ECM element hyaluronic acid and test formulations of insulin and four different monoclonal antibodies. Our findings suggest that Scissor can provide a tractable method to examine the potential fate of a biopharmaceutical formulation after its SC injection in humans and that this approach may provide a reliable and representative alternative to animal testing for the initial screening of SC formulations.
Asunto(s)
Productos Biológicos/administración & dosificación , Productos Biológicos/farmacocinética , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/farmacocinética , Bicarbonatos/química , Productos Biológicos/química , Tampones (Química) , Química Farmacéutica , Diálisis , Diseño de Fármacos , Matriz Extracelular/metabolismo , Humanos , Ácido Hialurónico/metabolismo , Inyecciones Subcutáneas , Linfa/metabolismo , Tejido Subcutáneo/metabolismoRESUMEN
PURPOSE: Trastuzumab-emtansine (T-DM1) is an antibody-drug conjugate (ADC) comprising the cytotoxic agent DM1 conjugated to trastuzumab with a stable linker. Thrombocytopenia was the dose-limiting toxicity in the phase I study, and grade ≥3 thrombocytopenia occurred in up to 13% of patients receiving T-DM1 in phase III studies. We investigated the mechanism of T-DM1-induced thrombocytopenia. EXPERIMENTAL DESIGN: The effect of T-DM1 on platelet function was measured by aggregometry, and by flow cytometry to detect the markers of activation. The effect of T-DM1 on differentiation and maturation of megakaryocytes (MK) from human hematopoietic stem cells was assessed by flow cytometry and microscopy. Binding, uptake, and catabolism of T-DM1 in MKs, were assessed by various techniques including fluorescence microscopy, scintigraphy to detect T-[H(3)]-DM1 and (125)I-T-DM1, and mass spectrometry. The role of FcγRIIa was assessed using blocking antibodies and mutant constructs of trastuzumab that do not bind FcγR. RESULTS: T-DM1 had no direct effect on platelet activation and aggregation, but it did markedly inhibit MK differentiation via a cytotoxic effect. Inhibition occurred with DM1-containing ADCs but not with trastuzumab demonstrating a role for DM1. MKs internalized these ADCs in a HER2-independent, FcγRIIa-dependent manner, resulting in intracellular release of DM1. Binding and internalization of T-DM1 diminished as MKs matured; however, prolonged exposure of mature MKs to T-DM1 resulted in a disrupted cytoskeletal structure. CONCLUSIONS: These data support the hypothesis that T-DM1-induced thrombocytopenia is mediated in large part by DM1-induced impairment of MK differentiation, with a less pronounced effect on mature MKs.
Asunto(s)
Anticuerpos Monoclonales Humanizados/efectos adversos , Antineoplásicos/efectos adversos , Neoplasias de la Mama/tratamiento farmacológico , Maitansina/análogos & derivados , Trombocitopenia/patología , Ado-Trastuzumab Emtansina , Anticuerpos Monoclonales Humanizados/administración & dosificación , Antineoplásicos/administración & dosificación , Neoplasias de la Mama/complicaciones , Neoplasias de la Mama/patología , Diferenciación Celular/efectos de los fármacos , Femenino , Humanos , Inmunoconjugados/administración & dosificación , Inmunoconjugados/efectos adversos , Maitansina/administración & dosificación , Maitansina/efectos adversos , Megacariocitos/efectos de los fármacos , Activación Plaquetaria/efectos de los fármacos , Plasma Rico en Plaquetas/efectos de los fármacos , Receptor ErbB-2/metabolismo , Trombocitopenia/inducido químicamente , Trombocitopenia/etiología , TrastuzumabRESUMEN
Streptococcus pneumoniae open reading frame SP0082 encodes a surface protein that contains four copies of a novel conserved repeat domain that bears no significant sequence similarity to proteins of known function. Homologous sequences from other streptococci contain two to six of these repeats, designated the SSURE (streptococcal surface repeat) domain. To investigate the functional role(s) of this domain, the third SSURE repeat of SP0082 sequence has been expressed in Escherichia coli, purified to homogeneity and characterized by biochemical and immunological methods. The expressed protein fragment was found to bind to fibronectin, but not to collagen or submaxillary mucin. Anti-SSURE antibodies recognized the corresponding protein on the surface of pneumococcal cells. These data identify S. pneumoniae SP0082 protein and its homologs in other streptococci as fibronectin-binding surface adhesins. The SSURE domain is likely to contain a novel protein fold, which was tentatively modeled using ab initio modeling methods.
Asunto(s)
Adhesión Bacteriana , Biología Computacional/métodos , Fibronectinas/química , Streptococcus pneumoniae/metabolismo , Streptococcus pneumoniae/patogenicidad , Adhesinas Bacterianas/química , Secuencia de Aminoácidos , Animales , Proteínas de la Membrana Bacteriana Externa/química , Proteínas Bacterianas/química , Membrana Celular/metabolismo , Clonación Molecular , Colágeno/química , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Fibronectinas/metabolismo , Citometría de Flujo , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Especificidad de la EspecieRESUMEN
Antibodies to transferrin receptor (TfR) have potential use for therapeutic entry into the brain. We have shown that bispecific antibodies against TfR and ß-secretase (BACE1 [ß-amyloid cleaving enzyme-1]) traverse the blood-brain barrier (BBB) and effectively reduce brain amyloid ß levels. We found that optimizing anti-TfR affinity improves brain exposure and BACE1 inhibition. Here we probe the cellular basis of this improvement and explore whether TfR antibody affinity alters the intracellular trafficking of TfR. Comparing high- and low-affinity TfR bispecific antibodies in vivo, we found that high-affinity binding to TfR caused a dose-dependent reduction of brain TfR levels. In vitro live imaging and colocalization experiments revealed that high-affinity TfR bispecific antibodies facilitated the trafficking of TfR to lysosomes and thus induced the degradation of TfR, an observation which was further confirmed in vivo. Importantly, high-affinity anti-TfR dosing induced reductions in brain TfR levels, which significantly decreased brain exposure to a second dose of low-affinity anti-TfR bispecific. Thus, high-affinity anti-TfR alters TfR trafficking, which dramatically impacts the capacity for TfR to mediate BBB transcytosis.
Asunto(s)
Anticuerpos Biespecíficos/metabolismo , Encéfalo/inmunología , Encéfalo/metabolismo , Receptores de Transferrina/inmunología , Receptores de Transferrina/metabolismo , Secretasas de la Proteína Precursora del Amiloide/inmunología , Animales , Afinidad de Anticuerpos , Ácido Aspártico Endopeptidasas/inmunología , Barrera Hematoencefálica/inmunología , Barrera Hematoencefálica/metabolismo , Femenino , Lisosomas/inmunología , Lisosomas/metabolismo , Ratones , Ratones Endogámicos C57BL , Transporte de Proteínas , Transcitosis/inmunología , Transcitosis/fisiologíaRESUMEN
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
Delta-like-4 ligand (DLL4) plays an important role in vascular development and is widely expressed on the vasculature of normal and tumor tissues. Anti-DLL4 is a humanized IgG1 monoclonal antibody against DLL4. The purpose of these studies was to characterize the pharmacokinetics (PK), tissue distribution, and anti-tumor efficacy of anti-DLL4 in mice over a range of doses. PK and tissue distribution of anti-DLL4 were determined in athymic nude mice after administration of single intravenous (IV) doses. In the tissue distribution study, radiolabeled anti-DLL4 (mixture of (125)Iodide and (111)Indium) was administered in the presence of increasing amounts of unlabeled anti-DLL4. Dose ranging anti-DLL4 anti-tumor efficacy was evaluated in athymic nude mice bearing MV522 human lung tumor xenografts. Anti-DLL4 had nonlinear PK in mice with rapid serum clearance at low doses and slower clearance at higher doses suggesting the involvement of target mediated clearance. Consistent with the PK data, anti-DLL4 was shown to specifically distribute to several normal tissues known to express DLL4 including the lung and liver. Maximal efficacy in the xenograft model was seen at doses ≥ 10 mg/kg when tissue sinks were presumably saturated, consistent with the PK and tissue distribution profiles. These findings highlight the importance of mechanistic understanding of antibody disposition to enable dosing strategies for maximizing efficacy.
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Anticuerpos Monoclonales Humanizados/farmacocinética , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/sangre , Anticuerpos Monoclonales Humanizados/inmunología , Área Bajo la Curva , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Radioisótopos de Indio/farmacocinética , Péptidos y Proteínas de Señalización Intracelular/inmunología , Radioisótopos de Yodo/farmacocinética , Neoplasias Pulmonares/inmunología , Proteínas de la Membrana/inmunología , Tasa de Depuración Metabólica , Ratones Desnudos , Distribución Tisular , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Using therapeutic antibodies that need to cross the blood-brain barrier (BBB) to treat neurological disease is a difficult challenge. We have shown that bispecific antibodies with optimized binding to the transferrin receptor (TfR) that target ß-secretase (BACE1) can cross the BBB and reduce brain amyloid-ß (Aß) in mice. Can TfR enhance antibody uptake in the primate brain? We describe two humanized TfR/BACE1 bispecific antibody variants. Using a human TfR knock-in mouse, we observed that anti-TfR/BACE1 antibodies could cross the BBB and reduce brain Aß in a TfR affinity-dependent fashion. Intravenous dosing of monkeys with anti-TfR/BACE1 antibodies also reduced Aß both in cerebral spinal fluid and in brain tissue, and the degree of reduction correlated with the brain concentration of anti-TfR/BACE1 antibody. These results demonstrate that the TfR bispecific antibody platform can robustly and safely deliver therapeutic antibody across the BBB in the primate brain.
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Secretasas de la Proteína Precursora del Amiloide/inmunología , Anticuerpos Biespecíficos/farmacocinética , Antígenos CD/inmunología , Ácido Aspártico Endopeptidasas/inmunología , Barrera Hematoencefálica/metabolismo , Permeabilidad Capilar , Receptores de Transferrina/inmunología , Administración Intravenosa , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/líquido cefalorraquídeo , Animales , Anticuerpos Biespecíficos/administración & dosificación , Anticuerpos Biespecíficos/sangre , Anticuerpos Biespecíficos/inmunología , Especificidad de Anticuerpos , Antígenos CD/genética , Antígenos CD/metabolismo , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Ácido Aspártico Endopeptidasas/metabolismo , Transporte Biológico , Células CHO , Cricetulus , Reacciones Cruzadas , Regulación hacia Abajo , Células HEK293 , Humanos , Macaca fascicularis , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/líquido cefalorraquídeo , Receptores de Transferrina/genética , Receptores de Transferrina/metabolismo , TransfecciónRESUMEN
A known limitation of iodine radionuclides for labeling and biological tracking of receptor targeted proteins is the tendency of iodotyrosine to rapidly diffuse from cells following endocytosis and lysosomal degradation. In contrast, radiometal-chelate complexes such as indium-111-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (In-111-DOTA) accumulate within target cells due to the residualizing properties of the polar, charged metal-chelate-amino acid adduct. Iodine radionuclides boast a diversity of nuclear properties and chemical means for incorporation, prompting efforts to covalently link radioiodine with residualizing molecules. Herein, we describe the Ugi-assisted synthesis of [I-125]HIP-DOTA, a 4-hydroxy-3-iodophenyl (HIP) derivative of DOTA, and demonstration of its residualizing properties in a murine xenograft model. Overall, this study displays the power of multicomponent synthesis to yield a versatile radioactive probe for antibodies across multiple therapeutic areas with potential applications in both preclinical biodistribution studies and clinical radioimmunotherapies.
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Anticuerpos Monoclonales de Origen Murino/metabolismo , Dipéptidos/síntesis química , Compuestos Heterocíclicos con 1 Anillo/síntesis química , Inmunoconjugados/química , Succinimidas/síntesis química , Animales , Anticuerpos Monoclonales de Origen Murino/química , Anticuerpos Monoclonales de Origen Murino/uso terapéutico , Complejos de Coordinación/metabolismo , Dipéptidos/metabolismo , Compuestos Heterocíclicos con 1 Anillo/metabolismo , Inmunoconjugados/metabolismo , Radioisótopos de Indio , Ratones , Radioinmunoterapia , Succinimidas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Monoclonal antibodies have provided many validated and potential new therapeutic candidates for various diseases encompassing the realms of neurology, ophthalmology, immunology, and especially oncology. The mechanism of action for these biological molecules typically involves specific binding to a soluble ligand or cell surface protein in order to block or alter a molecular pathway, induce a desired cellular response, or deplete a target cell. Many antigens reside within the interstitial space, the fluid-filled compartment that lies between the outer endothelial vessel wall and the plasma membranes of cells. This mini-review examines the concepts relevant to the kinetics and behavior of antibodies within the interstitium with a special emphasis on radiometric measurement of quantitative pharmacology. Molecular probes are discussed to outline chemical techniques, selection criteria, data interpretation, and relevance to the study of antibody pharmacokinetics. The importance of studying the tissue uptake of antibodies at a compartmental level is highlighted, including a brief overview of receptor occupancy and its interpretation in radiotracer studies. Experimental methods for measuring the spatial composition of tissues are examined in terms of relative vascular, interstitial, and cellular volumes using solid tumors as a representative example. Experimental methods and physiologically based pharmacokinetic modeling are introduced as distinct approaches to distinguish between free and bound fractions of interstitial antibody. Overall, the review outlines the available methods for pharmacokinetic measurements of antibodies and physiological measurements of the compartments that they occupy, while emphasizing that such approaches may not fully capture the complexities of dynamic, heterogeneous tumors and other tissues.
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Anticuerpos/metabolismo , Animales , Humanos , Distribución TisularRESUMEN
Monoclonal antibodies are increasingly being developed to treat multiple disease areas, including those related to oncology, immunology, neurology, and ophthalmology. There are multiple factors, such as charge, size, neonatal Fc receptor (FcRn) binding affinity, target affinity and biology, immunoglobulin G (IgG) subclass, degree and type of glycosylation, injection route, and injection site, that could affect the pharmacokinetics (PK) of these large macromolecular therapeutics, which in turn could have ramifications on their efficacy and safety. This minireview examines how characteristics of the antibodies could be altered to change their PK profiles. For example, it was observed that a net charge modification of at least a 1-unit shift in isoelectric point altered antibody clearance. Antibodies with enhanced affinity for FcRn at pH 6.0 display longer serum half-lives and slower clearances than wild type. Antibody fragments have different clearance rates and tissue distribution profiles than full length antibodies. Fc glycosylation is perceived to have a minimal effect on PK while that of terminal high mannose remains unclear. More investigation is warranted to determine if injection route and/or site impacts PK. Nonetheless, a better understanding of the effects of all these variations may allow for the better design of antibody therapeutics.