RESUMEN
CD70 is highly expressed in renal cell carcinoma (RCC), with limited expression in normal tissue, making it an attractive CAR T target for an immunogenic solid tumor indication. Here we generated and characterized a panel of anti-CD70 single-chain fragment variable (scFv)-based CAR T cells. Despite the expression of CD70 on T cells, production of CAR T cells from a subset of scFvs with potent in vitro activity was achieved. Expression of CD70 CARs masked CD70 detection in cis and provided protection from CD70 CAR T cell-mediated fratricide. Two distinct classes of CAR T cells were identified with differing memory phenotype, activation status, and cytotoxic activity. Epitope mapping revealed that the two classes of CARs bind unique regions of CD70. CD70 CAR T cells displayed robust antitumor activity against RCC cell lines and patient-derived xenograft mouse models. Tissue cross-reactivity studies identified membrane staining in lymphocytes, thus matching the known expression pattern of CD70. In a cynomolgus monkey CD3-CD70 bispecific toxicity study, expected findings related to T-cell activation and elimination of CD70-expressing cells were observed, including cytokine release and loss of cellularity in lymphoid tissues. Finally, highly functional CD70 allogeneic CAR T cells were produced at large scale through elimination of the T-cell receptor by TALEN-based gene editing. Taken together, these efficacy and safety data support the evaluation of CD70 CAR T cells for the treatment of RCC and has led to the advancement of an allogeneic CD70 CAR T-cell candidate into phase I clinical trials. SIGNIFICANCE: These findings demonstrate the efficacy and safety of fratricide-resistant, allogeneic anti-CD70 CAR T cells targeting renal cell carcinoma and the impact of CAR epitope on functional activity. See related commentary by Adotévi and Galaine, p. 2517.
Asunto(s)
Carcinoma de Células Renales , Trasplante de Células Madre Hematopoyéticas , Neoplasias Renales , Animales , Ligando CD27 , Carcinoma de Células Renales/patología , Línea Celular Tumoral , Humanos , Inmunoterapia Adoptiva , Neoplasias Renales/patología , Macaca fascicularis , Ratones , Linfocitos T/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
INTRODUCTION: The cardiovascular liability of candidate compounds can be evaluated by a number of methods including implanted telemetry, jacketed telemetry and surface lead electrocardiogram (ECG). The utility of the new PhysioTel™ Digital M11 cardiovascular telemetry implant was evaluated in monkeys and dogs. METHODS: Eight monkeys and dogs (4 males and 4 females per species) were implanted with the M11 device utilizing a femoral blood pressure catheter and periosteal ECG leads. The signal quality of the ECGs was determined as a percentage of software-matched waveforms and as a percentage of signal loss during the recording periods. To investigate sensitivity for detecting changes in QT/QTc and HR/BP, moxifloxacin and doxazosin were administered to monkeys and dogs implanted with the M11 device. Additionally, histopathological evaluation of the implant site was completed. RESULTS: For both monkey and dog, the percentage of recognizable waveforms was high (65% and 85%, respectively), while the average amount of signal loss was low (1% and 3%, respectively), indicating that the M11 implants delivered data of sufficient quality. In monkeys, moxifloxacin (90mg/kg) induced QT and QTc prolongation up to 22 and 12ms, respectively, while at 30mg/kg in dogs, the maximal increases in QT and QTc were 13 and 16ms, respectively. Doxazosin (1.5 and 1.0mg/kg) produced HR increases up to 35 and 29bpm with decreases in blood pressure up to -14 and -26mmHg in monkeys and dogs, respectively. The histopathological impact of the implant, catheter and biopotential leads was limited to expected minor local inflammatory changes as assessed at necropsy and with microscopic examination. DISCUSSION: Based upon the results of this study, the PhysioTel™ Digital M11 is a suitable technology for assessing cardiovascular parameters in monkeys and dogs, and because of the size and limited invasiveness of the implant, is well positioned for use on toxicology studies.
Asunto(s)
Sistema Cardiovascular/fisiopatología , Síndrome de QT Prolongado/fisiopatología , Telemetría/métodos , Animales , Presión Sanguínea/efectos de los fármacos , Presión Sanguínea/fisiología , Sistema Cardiovascular/efectos de los fármacos , Perros , Doxazosina/farmacología , Electrocardiografía/métodos , Femenino , Fluoroquinolonas/farmacología , Frecuencia Cardíaca/efectos de los fármacos , Frecuencia Cardíaca/fisiología , Inflamación/tratamiento farmacológico , Inflamación/fisiopatología , Síndrome de QT Prolongado/tratamiento farmacológico , Macaca fascicularis , Masculino , MoxifloxacinoRESUMEN
Two 6-month repeat-dose toxicity studies in cynomolgus monkeys illustrated immune complex-mediated adverse findings in individual monkeys and identified parameters that potentially signal the onset of immune complex-mediated reactions following administration of RN6G, a monoclonal antibody (mAb). In the first study, 3 monkeys exhibited nondose-dependent severe clinical signs accompanied by decreased erythrocytes with increased reticulocytes, neutrophilia, monocytosis, thrombocytopenia, coagulopathy, decreased albumin, azotemia, and increased serum levels of activated complement products, prompting unscheduled euthanasia. Histologically, immunohistochemical localization of RN6G was associated with monkey immunoglobulin and complement components in glomeruli and other tissues, attributable to immune complex disease (ICD). All 3 animals also had anti-RN6G antibodies and decreased plasma levels of RN6G. Subsequently, an investigational study was designed and conducted with regulatory agency input to detect early onset of ICD and assess reversibility to support further clinical development. Dosing of individual animals ceased when biomarkers of ICD indicated adverse findings. Of the 12 monkeys, 1 developed anti-RN6G antibodies and decreased RN6G exposure that preceded elevations in complement products, interleukin-6, and coagulation parameters and decreases in albumin and fibrinogen. All findings in this monkey, except for antidrug antibody (ADA), reversed after cessation of dosing without progressing to adverse sequelae typically associated with ICD.
Asunto(s)
Anticuerpos Monoclonales/efectos adversos , Biomarcadores/sangre , Efectos Colaterales y Reacciones Adversas Relacionados con Medicamentos , Enfermedades del Complejo Inmune/sangre , Animales , Anticuerpos Monoclonales/sangre , Proteína C-Reactiva/metabolismo , Complejo de Ataque a Membrana del Sistema Complemento/metabolismo , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Femenino , Enfermedades del Complejo Inmune/inducido químicamente , Enfermedades del Complejo Inmune/patología , Inmunohistoquímica , Interferón gamma/sangre , Interleucina-1beta/sangre , Interleucina-6/sangre , Macaca fascicularis , Masculino , Microscopía Electrónica de Transmisión , Factor de Necrosis Tumoral alfa/sangre , UrinálisisRESUMEN
Imatinib and bosutinib were administered to rats for up to 6 months at clinically relevant exposures to investigate the effects on the cardiovascular system. Imatinib treatment resulted in increased volume, wall thickness and mass suggesting a hypertrophic heart in male and female rats at one and fivefold clinical exposures, respectively. Bosutinib treatment resulted in milder cardiac hypertrophy in female rats only at fivefold clinical exposures. Analysis of excised hearts and cultured myocytes demonstrated increased expression of hypertrophic genes with imatinib or analogs, but not bosutinib or c-Abl RNAi treatment. The current dataset suggests that cardiovascular liability of imatinib and bosutinib are differentiated preclinically and c-Abl independent.
Asunto(s)
Compuestos de Anilina/efectos adversos , Benzamidas/efectos adversos , Sistema Cardiovascular/efectos de los fármacos , Nitrilos/efectos adversos , Piperazinas/efectos adversos , Pirimidinas/efectos adversos , Quinolinas/efectos adversos , Compuestos de Anilina/administración & dosificación , Compuestos de Anilina/farmacocinética , Animales , Benzamidas/administración & dosificación , Benzamidas/farmacocinética , Cardiomiopatía Hipertrófica/inducido químicamente , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/patología , Sistema Cardiovascular/patología , Ecocardiografía , Femenino , Expresión Génica , Técnicas de Silenciamiento del Gen , Corazón/efectos de los fármacos , Corazón/fisiopatología , Mesilato de Imatinib , Masculino , Miocardio/metabolismo , Miocardio/patología , Nitrilos/administración & dosificación , Nitrilos/farmacocinética , Tamaño de los Órganos , Piperazinas/administración & dosificación , Piperazinas/farmacocinética , Proteínas Proto-Oncogénicas c-abl/genética , Pirimidinas/administración & dosificación , Pirimidinas/farmacocinética , Quinolinas/administración & dosificación , Quinolinas/farmacocinética , Ratas , Factores SexualesRESUMEN
The fibroblast growth factor receptors (FGFR) play a major role in angiogenesis and are desirable targets for the development of therapeutics. Groups of Wistar Han rats were dosed orally once daily for 4 days with a small molecule pan-FGFR inhibitor (5mg/kg) or once daily for 6 days with a small molecule MEK inhibitor (3mg/kg). Serum phosphorous and FGF23 levels increased in all rats during the course of the study. Histologically, rats dosed with either drug exhibited multifocal, multiorgan soft tissue mineralization. Expression levels of the sodium phosphate transporter Npt2a and the vitamin D-metabolizing enzymes Cyp24a1 and Cyp27b1 were modulated in kidneys of animals dosed with the pan-FGFR inhibitor. Both inhibitors decreased ERK phosphorylation in the kidneys and inhibited FGF23-induced ERK phosphorylation in vitro in a dose-dependent manner. A separate cardiovascular outcome study was performed to monitor hemodynamics and cardiac structure and function of telemetered rats dosed with either the pan-FGFR inhibitor or MEK inhibitor for 3 days. Both compounds increased blood pressure (~+ 17 mmHg), decreased heart rate (~-75 bpm), and modulated echocardiography parameters. Our data suggest that inhibition of FGFR signaling following administration of either pan-FGFR inhibitor or MEK inhibitor interferes with the FGF23 pathway, predisposing animals to hyperphosphatemia and a tumoral calcinosis-like syndrome in rodents.
Asunto(s)
Sistema Cardiovascular/fisiopatología , Factores de Crecimiento de Fibroblastos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Transducción de Señal , Animales , Secuencia de Bases , Sistema Cardiovascular/metabolismo , Línea Celular , Cartilla de ADN , Factor-23 de Crecimiento de Fibroblastos , Humanos , Fosforilación , Ratas , Ratas WistarRESUMEN
The Bcr-abl tyrosine kinase inhibitor imatinib mesylate is the frontline therapy for chronic myeloid leukemia. Imatinib has been reported to cause congestive heart failure and left ventricular contractile dysfunction in patients and cardiomyopathy in rodents, findings proposed to be associated with its pharmacological activity. To investigate the specific role of Abelson oncogene 1 (c-Abl) in imatinib-induced cardiac toxicity, we performed targeted gene inhibition of c-Abl by RNA interference in neonatal cardiomyocytes (NCMs). Suppression of c-Abl did not lead to cytotoxicity or induction of endoplasmic reticulum (ER) stress. To further dis associate c-Abl from imatinib-induced cardiac toxicity, we designed imatinib structural analogs that do not have appreciable c-Abl inhibition in NCMs. The c-Abl inactive analogs induced cytotoxicity and ER stress, at similar or greater potencies and magnitudes as imatinib. Furthermore, combining c-Abl gene silencing with imatinib and analogs treatment did not significantly shift the cytotoxicity dose response curves. Imatinib and analogs were shown to accumulate in lysosomes, likely due to their physicochemical properties, and disrupt autophagy. The toxicity induced by imatinib and analogs can be rescued by bafilomycin A pretreatment, demonstrating the involvement of lysosomal accumulation in cardiac toxicity. The results from our studies strongly suggest that imatinib induces cardiomyocyte dysfunction through disruption of autophagy and induction of ER stress, independent of c-Abl inhibition.
Asunto(s)
Antineoplásicos/toxicidad , Corazón/efectos de los fármacos , Piperazinas/toxicidad , Proteínas Quinasas/metabolismo , Pirimidinas/toxicidad , Animales , Secuencia de Bases , Benzamidas , Cartilla de ADN , Mesilato de Imatinib , Interferencia de ARN , Ratas , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
The receptor tyrosine kinase receptor (RTK) signaling pathway, mesenchymal-epithelial transition factor (c-Met)/hepatocyte growth factor receptor (HGFR), has been implicated in oncogenesis and is a target of interest in cancer therapy. PF-04254644 is a potent and selective inhibitor of c-Met/HGFR. Wide ligand binding profiling of PF-04254644 revealed a potentially significant interaction with phosphodiesterase (PDE) 3, and follow-up PDE enzyme activity assays confirmed PF-04254644 as a potent inhibitor of PDE3 as well as other PDEs (1, 2, 5, 10, and 11). Clinical observations, laboratory, and echocardiography parameters were recorded in Sprague-Dawley (SD) rats that received PF-04254644 oral dosing for up to seven consecutive days. Toxicological evaluations revealed myocardial degeneration as an adverse event at all tested doses. Echocardiographic evaluations revealed an increase in heart rate (HR) and contractility after the first dose with PF-04254644 and myocardial fibrosis correlated with decreased cardiac function after repeat dosing. A study in telemetry-instrumented rats substantiated that PF-04254644 induced a sustained increased HR and decreased contractility after six days of treatment. Data suggest that the decreased cardiac function and cardiotoxicity are likely due to inhibition of multiple PDEs by PF-04254644.
Asunto(s)
Corazón/efectos de los fármacos , Miocardio/patología , Hidrolasas Diéster Fosfóricas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/toxicidad , Quinolinas/toxicidad , Animales , Frecuencia Cardíaca/efectos de los fármacos , Masculino , Contracción Miocárdica/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Peroxisome-proliferator-activated receptor (PPAR-gamma) agonists improve insulin sensitivity, but are associated with edema. Increased distal tubule sodium and water reabsorption through the epithelial sodium channel (ENaC) and aquaporin-2 (AQP-2) have been suggested to play mechanistic roles. To determine the molecular regulation of these proteins, we treated male, Sprague-Dawley rats daily by gavage with either vehicle, rosiglitazone (RGZ, 50mg/kg bw), or PD168 (a test compound causing marked edema, 10mg/kg bw), for 1, 3, or 5 days (n=6/treatment/time). On day 1, urine sodium excretion was significantly reduced by RGZ with a strong trend for PD168 (p-values 0.047 and 0.053, respectively) indicating early sodium retention. Blood pressure was lowered by RGZ- or PD168 treatment by 12h. Immunoblotting of whole kidney homogenates (WKHs) and a membrane-enriched fraction (MF) revealed increased band densities for AQP-2 in WKH (29 kDa and glycosylated bands) by both drugs at 1 day. However, at 5 days, the 29-kDa band was significantly decreased ( approximately 30% of vehicle). alpha-ENaC was increased by RGZ at 3 days; however both agents decreased alpha-ENaC by 5 days. In contrast, beta- and gamma-ENaC (85 kDa) were unchanged or decreased at all times by both agents. However, the 70-kDa band of gamma-ENaC (active band) in MF was increased in density (120-600%) by both agents on days 3-5. Overall, both agents resulted in early alterations in banding patterns for AQP-2 and ENaC subunits, many of which are described as activating changes. However, later reduction in AQP-2 and alpha-ENaC may represent an attempt to re-establish sodium and water balance.
Asunto(s)
Acuaporina 2/metabolismo , Edema/inducido químicamente , Edema/metabolismo , Canales Epiteliales de Sodio/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Oxazoles/toxicidad , PPAR gamma/agonistas , Triazoles/toxicidad , Adiponectina/sangre , Animales , Presión Sanguínea/efectos de los fármacos , Insulina/sangre , Masculino , Natriuresis/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Rosiglitazona , Tiazolidinedionas/toxicidad , Equilibrio Hidroelectrolítico/efectos de los fármacosRESUMEN
Proinflammatory cytokines inhibit learning and memory but the significance of interleukin-6 (IL-6) in acute cognitive deficits induced by the peripheral innate immune system is not known. To examine the functional role of IL-6 in hippocampus-mediated cognitive impairments associated with peripheral infections, C57BL6/J (IL-6(+/+)) and IL-6 knock-out (IL-6(-/-)) mice were trained in a matching-to-place version of the water maze. After an acquisition phase, IL-6(+/+) mice injected intraperitoneally with lipopolysaccharide (LPS) exhibited deficits in working memory. However, IL-6(-/-) mice were refractory to the LPS-induced impairment in working memory. To determine the mechanism by which IL-6 deficiency conferred protection from disruption in working memory, plasma IL-1beta and tumor necrosis factor alpha (TNFalpha), c-Fos immunoreactivity in the nucleus of the solitary tract (NTS), and steady-state levels of IL-1beta and TNFalpha mRNA in neuronal layers of the hippocampus were determined in IL-6(+/+) and IL-6(-/-) mice after injection of LPS. Plasma IL-1beta and TNFalpha and c-Fos immunoreactivity in the NTS were increased similarly in IL-6(+/+) and IL-6(-/-) mice after LPS, indicating high circulating levels of IL-1beta and TNFalpha and activation of vagal afferent pathways were not sufficient to disrupt working memory in the absence of IL-6. However, the LPS-induced upregulation of IL-1beta and TNFalpha mRNA that was evident in hippocampal tissue of IL-6(+/+) mice was greatly attenuated or entirely absent in IL-6(-/-) mice. Collectively, these data suggest that humoral and neural immune-to-brain communication pathways are intact in IL-6-deficient mice but that, in the absence of IL-6, the central cytokine compartment is hyporesponsive.
Asunto(s)
Citocinas/biosíntesis , Hipocampo/metabolismo , Interleucina-6/fisiología , Lipopolisacáridos/toxicidad , Memoria/fisiología , Neuronas/metabolismo , Animales , Citocinas/genética , Regulación de la Expresión Génica/inmunología , Mediadores de Inflamación/metabolismo , Mediadores de Inflamación/fisiología , Interleucina-6/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas/inmunologíaRESUMEN
Heart failure is a complex multifactorial disease resulting in a myriad of progressive changes at the molecular, cellular, and physiological level. To better understand the mechanisms associated with the development of congestive heart failure, a comprehensive examination of the aging lean male spontaneously hypertensive, heart failure-prone rat (SHHF) was conducted. Myocardial function and structural integrity progressively diminished as evidenced by decreased ejection fraction and increased left ventricular volume measured using echocardiography. Functional and structural changes were accompanied by elevations in circulating inflammatory markers, including tumor necrosis factor-alpha (TNF-alpha), IL-6, and TNF receptors type 1 and 2. Increased systemic inflammatory marker levels were consistent with age-dependent changes in the expression pattern of genes that contribute to stress, inflammation, and the extracellular matrix in SHHF animals analyzed from age 4 to 18 mo. In summary, the SHHF rat shares many hallmark features of the human disease state and represents a key experimental model for the dissection of complex human heart failure pathophysiology.