RESUMEN
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
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
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
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
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
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.
Asunto(s)
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.
Asunto(s)
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 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 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
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
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.
Asunto(s)
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
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
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.
Asunto(s)
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.
Asunto(s)
Anticuerpos/uso terapéutico , Glicosilación , Punto IsoeléctricoRESUMEN
Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate in clinical development for the treatment of human epidermal growth factor receptor 2 (HER2)-positive cancers. Herein, we describe a series of studies to assess T-DM1 absorption, distribution, metabolism, and excretion (ADME) in rats as well as to assess human exposure to T-DM1 catabolites. Following administration of unlabeled and radiolabeled T-DM1 in female Sprague Dawley rats as a single dose, plasma, urine, bile and feces were assessed for mass balance, profiling and identification of catabolites. In rats, the major circulating species in plasma was T-DM1, while DM1 concentrations were low (1.08 to 15.6 ng/mL). The major catabolites found circulating in rat plasma were DM1, [N-maleimidomethyl] cyclohexane-1- carboxylate-DM1 (MCC-DM1), and Lysine-MCC-DM1. These catabolites identified in rats were also detected in plasma samples from patients with HER2-positive metastatic breast cancer who received single-agent T-DM1 (3.6 mg/kg every 3 weeks) in a phase 2 clinical study. There was no evidence of tissue accumulation in rats or catabolite accumulation in human plasma following multiple dosing. In rats, T-DM1 was distributed nonspecifically to the organs without accumulation. The major pathway of DM1-containing catabolite elimination in rats was the fecal/biliary route, with up to 80% of radioactivity recovered in the feces and 50% in the bile. The rat T-DM1 ADME profile is likely similar to the human profile, although there may be differences since trastuzumab does not bind the rat HER2- like receptor. Further research is necessary to more fully understand the T-DM1 ADME profile in humans.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Anticuerpos Monoclonales Humanizados/farmacocinética , Maitansina/análogos & derivados , Ado-Trastuzumab Emtansina , Animales , Anticuerpos Monoclonales Humanizados/metabolismo , Evaluación Preclínica de Medicamentos/métodos , Femenino , Maitansina/química , Maitansina/metabolismo , Maitansina/farmacocinética , Metabolismo/efectos de los fármacos , Metabolismo/fisiología , Ratas , Ratas Sprague-Dawley , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiología , TrastuzumabRESUMEN
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
BACKGROUND: In evaluating the serum concentrations in mice of a Sema3E IgG1 Fc fusion protein, a possible antitumor agent, two ELISAs were developed: a generic assay detecting only the Fc portion of the therapeutic and a specific receptor-binding assay detecting intact protein. RESULTS: An unexpected discrepancy was observed in the measured in vivo serum concentrations, with the generic ELISA yielding higher concentrations than the specific ELISA. Size-exclusion HPLC and SDS-PAGE analysis of in vitro serum stability samples revealed extensive aggregation of Sema3E-Fc. The generic assay recovered more Sema3E-Fc in the presence of aggregates than the specific assay. CONCLUSION: Biophysical characterization combined with immunochemical analysis was key to elucidating not only the nature of the protein instability, but also the cause for the assay discrepancy.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática , Fragmentos Fc de Inmunoglobulinas/metabolismo , Proteínas Recombinantes de Fusión/sangre , Semaforinas/metabolismo , Animales , Anticuerpos/inmunología , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Femenino , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/inmunología , Ratones , Estabilidad Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Semaforinas/genética , Semaforinas/inmunología , Factores de TiempoRESUMEN
Both human epidermal growth factor receptor 2 (HER-2/neu) and VEGF overexpression correlate with aggressive phenotypes and decreased survival among breast cancer patients. Concordantly, the combination of trastuzumab (anti-HER2) with bevacizumab (anti-VEGF) has shown promising results in preclinical xenograft studies and in clinical trials. However, despite the known antiangiogenic mechanism of anti-VEGF antibodies, relatively little is known about their effects on the pharmacokinetics and tissue distribution of other antibodies. This study aimed to measure the disposition properties, with a particular emphasis on tumor uptake, of trastuzumab in the presence or absence of anti-VEGF. Radiolabeled trastuzumab was administered alone or in combination with an anti-VEGF antibody to mice bearing HER2-expressing KPL-4 breast cancer xenografts. Biodistribution, autoradiography, and single-photon emission computed tomography-X-ray computed tomography imaging all showed that anti-VEGF administration reduced accumulation of trastuzumab in tumors despite comparable blood exposures and similar distributions in most other tissues. A similar trend was also observed for an isotype-matched IgG with no affinity for HER2, showing reduced vascular permeability to macromolecules. Reduced tumor blood flow (P < 0.05) was observed following anti-VEGF treatment, with no significant differences in the other physiologic parameters measured despite immunohistochemical evidence of reduced vascular density. In conclusion, anti-VEGF preadministration decreased tumor uptake of trastuzumab, and this phenomenon was mechanistically attributed to reduced vascular permeability and blood perfusion. These findings may ultimately help inform dosing strategies to achieve improved clinical outcomes.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Monoclonales/farmacología , Neoplasias de la Mama/tratamiento farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales Humanizados/química , Afinidad de Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunohistoquímica , Radioisótopos de Indio/química , Radioisótopos de Indio/farmacocinética , Radioisótopos de Yodo/química , Radioisótopos de Yodo/farmacocinética , Ratones , Ratones Desnudos , Imagen Multimodal , Tomografía de Emisión de Positrones , Receptor ErbB-2/inmunología , Receptor ErbB-2/metabolismo , Distribución Tisular , Tomografía Computarizada por Rayos X , Trastuzumab , Factor A de Crecimiento Endotelial Vascular/inmunología , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A majority of human therapeutic antibody candidates show pharmacokinetic properties suitable for clinical use, but an unexpectedly fast antibody clearance is sometimes observed that may limit the clinical utility. Pharmacokinetic data in cynomolgus monkeys collected for a panel of 52 antibodies showed broad distribution of target-independent clearance values (2.4-61.3 mL/day/kg), with 15 (29%) having clearance > 10 mL/day/kg. Alteration in the interaction with the recycling FcRn receptor did not account for the faster than expected clearance observed for the antibodies; off-target binding was presumed to account for the fast clearance. We developed an assay based on ELISA detection of non-specific binding to baculovirus particles that can identify antibodies having increased risk for fast clearance. This assay can be used during lead generation or optimization to identify antibodies with increased risk of having fast clearance in both humans and cynomolgus monkeys, and thus increase the likelihood of obtaining a suitable drug candidate.