RESUMEN
BACKGROUND: Teclistamab (JNJ-64007957), a B-cell maturation antigen × CD3 bispecific antibody, displayed potent T-cell-mediated cytotoxicity of multiple myeloma cells in preclinical studies. OBJECTIVE: A first-in-human, Phase I, dose escalation study (MajesTEC-1) is evaluating teclistamab in patients with relapsed/refractory multiple myeloma. PATIENTS AND METHODS: To estimate the efficacious therapeutic dosing range of teclistamab, pharmacokinetic (PK) data following the first cycle doses in the low-dose cohorts in the Phase I study were modeled using a 2-compartment model and simulated to predict the doses that would have average and trough serum teclistamab concentrations in the expected therapeutic range (between EC50 and EC90 values from an ex vivo cytotoxicity assay). RESULTS: The doses predicted to have average serum concentrations between the EC50 and EC90 range were validated. In addition, simulations showed that weekly intravenous and subcutaneous doses of 0.70 mg/kg and 0.72 mg/kg, respectively, resulted in mean trough levels comparable to the maximum EC90. The most active doses in the Phase I study were weekly intravenous doses of 0.27 and 0.72 mg/kg and weekly subcutaneous doses of 0.72 and 1.5 mg/kg, with the weekly 1.5 mg/kg subcutaneous doses selected as the recommended Phase II dose (RP2D). With active doses, exposure was maintained above the mean EC90. All patients who responded to the RP2D of teclistamab had exposure above the maximum EC90 in both serum and bone marrow on cycle 3, Day 1 of treatment. CONCLUSIONS: Our findings show that PK simulations of early clinical data together with ex vivo cytotoxicity estimates can inform the identification of a bispecific antibody's therapeutic range. CLINICAL TRIAL REGISTRATION: NCT03145181, date of registration: May 9, 2017.
Asunto(s)
Antineoplásicos , Mieloma Múltiple , Administración Intravenosa , Antineoplásicos/uso terapéutico , Antígeno de Maduración de Linfocitos B , Humanos , Mieloma Múltiple/tratamiento farmacológicoRESUMEN
T-cell-mediated approaches have shown promise in myeloma treatment. However, there are currently a limited number of specific myeloma antigens that can be targeted, and multiple myeloma (MM) remains an incurable disease. G-protein-coupled receptor class 5 member D (GPRC5D) is expressed in MM and smoldering MM patient plasma cells. Here, we demonstrate that GPRC5D protein is present on the surface of MM cells and describe JNJ-64407564, a GPRC5DxCD3 bispecific antibody that recruits CD3+ T cells to GPRC5D+ MM cells and induces killing of GPRC5D+ cells. In vitro, JNJ-64407564 induced specific cytotoxicity of GPRC5D+ cells with concomitant T-cell activation and also killed plasma cells in MM patient samples ex vivo. JNJ-64407564 can recruit T cells and induce tumor regression in GPRC5D+ MM murine models, which coincide with T-cell infiltration at the tumor site. This antibody is also able to induce cytotoxicity of patient primary MM cells from bone marrow, which is the natural site of this disease. GPRC5D is a promising surface antigen for MM immunotherapy, and JNJ-64407564 is currently being evaluated in a phase 1 clinical trial in patients with relapsed or refractory MM (NCT03399799).
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos Inmunológicos/uso terapéutico , Mieloma Múltiple/terapia , Receptores Acoplados a Proteínas G/inmunología , Linfocitos T/efectos de los fármacos , Animales , Anticuerpos Biespecíficos/inmunología , Antineoplásicos Inmunológicos/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica/efectos de los fármacos , Femenino , Humanos , Inmunoterapia , Ratones Endogámicos BALB C , Mieloma Múltiple/inmunología , Linfocitos T/inmunologíaRESUMEN
Antibody-drug conjugates (ADC) represent a promising therapeutic modality for managing cancer. Here, we report a novel humanized ADC that targets the tetraspanin-like protein TM4SF1. TM4SF1 is highly expressed on the plasma membranes of many human cancer cells and also on the endothelial cells lining tumor blood vessels. TM4SF1 is internalized upon interaction with antibodies. We hypothesized that an ADC against TM4SF1 would inhibit cancer growth directly by killing cancer cells and indirectly by attacking the tumor vasculature. We generated a humanized anti-human TM4SF1 monoclonal antibody, v1.10, and armed it with an auristatin cytotoxic agent LP2 (chemical name mc-3377). v1.10-LP2 selectively killed cultured human tumor cell lines and human endothelial cells that express TM4SF1. Acting as a single agent, v1.10-LP2 induced complete regression of several TM4SF1-expressing tumor xenografts in nude mice, including non-small cell lung cancer and pancreas, prostate, and colon cancers. As v1.10 did not react with mouse TM4SF1, it could not target the mouse tumor vasculature. Therefore, we generated a surrogate anti-mouse TM4SF1 antibody, 2A7A, and conjugated it to LP2. At 3 mpk, 2A7A-LP2 regressed several tumor xenografts without noticeable toxicity. Combination therapy with v1.10-LP2 and 2A7A-LP2 together was more effective than either ADC alone. These data provide proof-of-concept that TM4SF1-targeting ADCs have potential as anticancer agents with dual action against tumor cells and the tumor vasculature. Such agents could offer exceptional therapeutic value and warrant further investigation. Mol Cancer Ther; 14(8); 1868-76. ©2015 AACR.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Proteínas de Neoplasias/antagonistas & inhibidores , Inhibidores de la Angiogénesis/toxicidad , Animales , Antígenos de Superficie/genética , Antígenos de Superficie/metabolismo , Antineoplásicos/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Células Endoteliales/efectos de los fármacos , Células Endoteliales/metabolismo , Expresión Génica , Humanos , Ratones , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Neovascularización Patológica , Conejos , Distribución Tisular , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Adiponectin overexpression in mice increases insulin sensitivity independent of adiposity. Here, we combined stable isotope infusion and in vivo measurements of lipid flux with transcriptomic analysis to characterize fatty acid metabolism in transgenic mice that overexpress adiponectin via the aP2-promoter (ADNTg). Compared with controls, fasted ADNTg mice demonstrated a 31% reduction in plasma free fatty acid concentrations (P = 0.008), a doubling of ketones (P = 0.028), and a 68% increase in free fatty acid turnover in plasma (15.1 ± 1.5 vs. 25.3 ± 6.8 mg/kg · min, P = 0.011). ADNTg mice had 2-fold more brown adipose tissue mass, and triglyceride synthesis and turnover were 5-fold greater in this organ (P = 0.046). Epididymal white adipose tissue was slightly reduced, possibly due to the approximately 1.5-fold increase in the expression of genes involved in oxidation (peroxisome proliferator-activated receptor α, peroxisome proliferator-activated receptor-γ coactivator 1α, and uncoupling protein 3). In ADNTg liver, lipogenic gene expression was reduced, but there was an unexpected increase in the expression of retinoid pathway genes (hepatic retinol binding protein 1 and retinoic acid receptor beta and adipose Cyp26A1) and liver retinyl ester content (64% higher, P < 0.02). Combined, these data support a physiological link between adiponectin signaling and increased efficiency of triglyceride synthesis and hydrolysis, a process that can be controlled by retinoids. Interactions between adiponectin and retinoids may underlie adiponectin's effects on intermediary metabolism.
Asunto(s)
Ácidos Grasos/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animales , Expresión Génica , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retinoides/metabolismo , Transducción de Señal , Triglicéridos/metabolismo , Vitamina A/análogos & derivados , Vitamina A/metabolismoRESUMEN
In this study, we elucidated the mechanism by which adiponectin modulates hepatic stellate cell activation and fibrogenesis. Adiponectin-overexpressing transgenic mice receiving thioacetamide were resistant to fibrosis, compared with controls. In contrast, adiponectin-null animals developed severe fibrosis. Expression of collagen α1(I) and α-smooth muscle actin (α-SMA) mRNAs were significantly lower in adiponectin-overexpressing mice, compared with controls. In wild-type stellate cells exposed to a lentivirus encoding adiponectin, expression of peroxisome proliferator-activated receptor-γ (PPARγ), SREBP1c, and CEBPα mRNAs was significantly increased (3.2-, 4.1-, and 2.2-fold, respectively; n = 3; P < 0.05, adiponectin virus versus control), consistent with possible activation of an adipogenic transcriptional program. Troglitazone, a PPARγ agonist, strongly suppressed up-regulation of collagen α1(I) and α-SMA mRNA in stellate cells isolated from wild-type mice; however, stellate cells from adiponectin-null animals failed to respond to troglitazone. Furthermore, in isolated stellate cells in which PPARγ was depleted using an adenovirus-Cre-recombinase system and in which adiponectin was also overexpressed, collagen α1(I) and α-SMA were significantly inhibited. We conclude that the PPARγ effect on stellate cell activation and the fibrogenic cascade appears to be adiponectin-dependent; however, the inhibitory effect of adiponectin on stellate cell activation was not dependent on PPARγ, suggesting the presence of PPARγ-dependent as well as independent pathways in stellate cells.
Asunto(s)
Células Estrelladas Hepáticas/metabolismo , PPAR gamma/metabolismo , Adiponectina/deficiencia , Adiponectina/metabolismo , Alanina Transaminasa/sangre , Animales , Aspartato Aminotransferasas/sangre , Peso Corporal , Susceptibilidad a Enfermedades , Eliminación de Gen , Células Estrelladas Hepáticas/patología , Ligandos , Hígado/metabolismo , Hígado/patología , Cirrosis Hepática/sangre , Cirrosis Hepática/metabolismo , Cirrosis Hepática/patología , Ratones , Ratones Transgénicos , Fenotipo , TioacetamidaRESUMEN
Obesity is an established risk factor in the pathogenesis of insulin resistance, type 2 diabetes mellitus and cardiovascular disease; all components that are part of the metabolic syndrome. Traditionally, insulin resistance has been defined in a glucocentric perspective. However, elevated systemic levels of fatty acids are now considered significant contributors towards the pathophysiological aspects associated with the syndrome. An overaccumulation of unoxidized long-chain fatty acids can saturate the storage capacity of adipose tissue, resulting in a lipid 'spill over' to non-adipose tissues, such as the liver, muscle, heart, and pancreatic-islets. Under these circumstances, such ectopic lipid deposition can have deleterious effects. The excess lipids are driven into alternative non-oxidative pathways, which result in the formation of reactive lipid moieties that promote metabolically relevant cellular dysfunction (lipotoxicity) and programmed cell-death (lipoapoptosis). Here, we focus on how both of these processes affect metabolically significant cell-types and highlight how lipotoxicity and sequential lipoapoptosis are as major mediators of insulin resistance, diabetes and cardiovascular disease.
Asunto(s)
Apoptosis , Diabetes Mellitus/fisiopatología , Lípidos/toxicidad , Animales , Diabetes Mellitus/metabolismo , Humanos , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiopatología , Metabolismo de los Lípidos , Hígado/fisiopatologíaRESUMEN
Adiponectin is a secretory protein predominantly expressed by adipocytes and released at a high rate into circulation. The ease with which the levels of adiponectin can be measured owing to its high abundance, small diurnal variation and high stability in plasma have made it a popular target for measurements in many clinical studies. It has emerged as a valuable biomarker for insulin sensitivity, cardiovascular risk and inflammation. However, adiponectin levels have been measured in many additional disease states. Preclinical studies not only have implicated adiponectin as an outstanding biomarker but also have demonstrated direct cardio-protective and insulin-sensitizing properties to be associated with the protein. Adiponectin might, therefore, be a viable protein therapeutic that could be supplied in a recombinant form in the context of type 2 diabetes and cardiovascular disease. However, the high abundance, complex tertiary and quaternary structure and rapid turnover might make chronic administration of the protein rather challenging.
Asunto(s)
Adiponectina/fisiología , Adiponectina/uso terapéutico , Adiponectina/metabolismo , Animales , Biomarcadores/metabolismo , Enfermedades Cardiovasculares/tratamiento farmacológico , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/fisiopatología , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas , Glucosa/metabolismo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Inflamación/fisiopatología , Resistencia a la Insulina/fisiología , Enfermedades Renales/metabolismo , Modelos Moleculares , Osteogénesis/fisiología , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
OBJECTIVE: The high-density lipoprotein (HDL) receptor scavenger receptor Class B type I (SR-BI) plays a key role in mediating the final step of reverse cholesterol transport. This study examined the possible regulation of hepatic SR-BI by phosphatidylinositol-3-kinase (PI3K), a well known regulator of endocytosis and membrane protein trafficking. METHODS AND RESULTS: SR-BI-dependent HDL selective cholesterol ester uptake in human HepG2 hepatoma cells was decreased (approximately 50%) by the PI3K inhibitors wortmannin and LY294002. Insulin increased selective uptake (approximately 30%), and this increase was blocked by PI3K inhibitors. Changes in SR-BI activity could be accounted for by pronounced changes in the subcellular localization and cell surface expression of SR-BI as determined by HDL cell surface binding, receptor biotinylation studies, and confocal fluorescence microscopy of HepG2 cells expressing green fluorescent protein-tagged SR-BI. Thus, under conditions of PI3K activation by insulin, and to a lesser extent by the SR-BI ligand HDL, cell surface expression of SR-BI was promoted, resulting in increased SR-BI-mediated HDL selective lipid uptake. CONCLUSIONS: Our data indicate that PI3K activation stimulates hepatic SR-BI function post-translationally by regulating the subcellular localization of SR-BI in a P13K-dependent manner. Decreased hepatocyte PI3K activity in insulin-resistant states, such as type 2 diabetes, obesity, or metabolic syndrome, may impair reverse cholesterol transport by reducing cell surface expression of SR-BI.
Asunto(s)
Hepatocitos/metabolismo , Metabolismo de los Lípidos/fisiología , Fosfatidilinositol 3-Quinasas/fisiología , Receptores Depuradores de Clase B/metabolismo , Fracciones Subcelulares/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Humanos , Insulina/farmacología , Lipoproteínas HDL/farmacología , Receptores Depuradores de Clase B/clasificación , Distribución Tisular/efectos de los fármacos , Distribución Tisular/fisiologíaRESUMEN
Previous studies have suggested that HDL retroendocytosis may play a role in scavenger receptor class B type I (SR-BI)-dependent selective lipid uptake in a cell-specific manner. To investigate this possibility, we developed methods to quantitatively measure HDL uptake and resecretion in fibroblast (COS-7) and hepatocyte (HepG2) cells expressing exogenous SR-BI. Approximately 17% and 24% of HDL associated in an SR-BI-dependent manner with COS-7 and HepG2 cells, respectively, accumulates intracellularly after a 10 min incubation. To determine whether this intracellular HDL undergoes retroendocytosis, we developed a pulse-chase assay whereby internalized biotinylated (125)I-HDL(3) secreted from cells is quantitatively precipitated from cell supernatants using immobilized streptavidin. Our results show a rapid secretion of a portion of intracellular HDL from both cell types (representing 4-7% of the total cell-associated HDL) that is almost complete within 30 min (half-life approximately 10 min). In COS-7 cells, the calculated rate of HDL secretion ( approximately 0.5 ng HDL/mg/min) was >30-fold slower than the rate of SR-BI-dependent selective cholesteryl ester (CE) uptake ( approximately 17 ng HDL/mg/min), whereas the rate of release of HDL from the cell surface ( approximately 19 ng HDL/mg/min) was similar to the rate of selective CE uptake. Notably, the rate of SR-BI-dependent HDL resecretion in COS-7 and HepG2 cells was similar. BLT1, a compound that inhibits selective CE uptake, does not alter the amount of SR-BI-mediated HDL retroendocytosis in COS-7 cells. From these data, we conclude that HDL retroendocytosis in COS-7 and HepG2 cells is similar and that the vast majority of SR-BI-dependent selective uptake occurs at the cell surface in both cell types.
Asunto(s)
Endocitosis/fisiología , Fibroblastos/metabolismo , Hepatocitos/metabolismo , Lipoproteínas HDL/metabolismo , Receptores Depuradores de Clase B/metabolismo , Animales , Biotinilación , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , Ésteres del Colesterol/metabolismo , Humanos , Metabolismo de los Lípidos , Ratones , Microscopía Confocal , Receptores Depuradores de Clase B/genética , Receptores Depuradores de Clase B/fisiologíaRESUMEN
The high density lipoprotein (HDL) receptor Scavenger Receptor BII (SR-BII) is encoded by an alternatively spliced mRNA from the SR-BI gene and is expressed in various tissues. SR-BII protein differs from SR-BI only in the carboxyl-terminal cytoplasmic tail, which, as we showed previously, must contain a signal that confers predominant intracellular expression and rapid endocytosis of HDL. We have shown that SR-BII mediates HDL endocytosis through aclathrin-dependent, caveolae-independent pathway. Two candidate amino acid motifs were identified in the tail that could mediate association with clathrin-containing endocytic vesicles: a putative dileucine motif at position 492-493 and an overlapping tyrosine-based YXXZ motif starting at position 489. Although substitution of tyrosine at position 489 with alanine or histidine did not affect endocytosis, substitution L492A resulted in increased surface binding of HDL and reduced HDL particle endocytosis. Substitution L493A had a less dramatic effect. No other regions in the carboxyl-terminal tail appeared to contain motifs required for HDL endocytosis. Substitutions of leucine at position 492 with the hydrophobic amino acids valine or phenylalanine also reduced HDL endocytosis, stressing the importance of leucine at this position. Introducing the SR-BII YTPLL motif into the carboxyl-terminal cytoplasmic tail of SR-BI converted SR-BI into an endocytic receptor resembling SR-BII. These results demonstrated that SR-BII differs from SR-BI in subcellular localization and trafficking and suggest that the two isoforms differ in the manner in which they target ligands intracellularly.