RESUMEN
T cells expressing CD19-targeting chimeric antigen receptors (CARs) reveal high efficacy in the treatment of B cell malignancies. Here, we report that T cell receptor fusion constructs (TRuCs) comprising an antibody-based binding domain fused to T cell receptor (TCR) subunits can effectively reprogram an intact TCR complex to recognize tumor surface antigens. Unlike CARs, TRuCs become a functional component of the TCR complex. TRuC-T cells kill tumor cells as potently as second-generation CAR-T cells, but at significant lower cytokine release and despite the absence of an extra co-stimulatory domain. TRuC-T cells demonstrate potent anti-tumor activity in both liquid and solid tumor xenograft models. In several models, TRuC-T cells are more efficacious than respective CAR-T cells. TRuC-T cells are shown to engage the signaling capacity of the entire TCR complex in an HLA-independent manner.
Asunto(s)
Inmunoterapia Adoptiva/métodos , Neoplasias/terapia , Receptores de Antígenos de Linfocitos T/inmunología , Receptores Artificiales/inmunología , Anticuerpos de Cadena Única/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD19/inmunología , Antígenos de Neoplasias/inmunología , Línea Celular Tumoral , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Neoplasias/inmunología , Cultivo Primario de Células , Dominios Proteicos , Receptores de Antígenos de Linfocitos T/genética , Receptores Artificiales/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Anticuerpos de Cadena Única/genética , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Cancer associated cachexia affects the majority of cancer patients during the course of the disease and thought to be directly responsible for about a quarter of all cancer deaths. Current evidence suggests that a pro-inflammatory state may be associated with this syndrome although the molecular mechanisms responsible for the development of cachexia are poorly understood. The purpose of this work was the identification of key drivers of cancer cachexia that could provide a potential point of intervention for the treatment and/or prevention of this syndrome. METHODS: Genetically engineered and xenograft tumour models were used to dissect the molecular mechanisms driving cancer cachexia. Cytokine profiling from the plasma of cachectic and non-cachectic cancer patients and mouse models was utilized to correlate circulating cytokine levels with the cachexia phenotype. RESULTS: Utilizing engineered tumour models we identified MAP3K11/GDF15 pathway activation as a potent inducer of cancer cachexia. Increased expression and high circulating levels of GDF15 acted as a key mediator of this process. In animal models, tumour-produced GDF15 was sufficient to trigger the cachexia phenotype. Elevated GDF15 circulating levels correlated with the onset and progression of cachexia in animal models and in patients with cancer. Inhibition of GDF15 biological activity with a specific antibody reversed body weight loss and restored muscle and fat tissue mass in several cachectic animal models regardless of their complex secreted cytokine profile. CONCLUSIONS: The combination of correlative observations, gain of function, and loss of function experiments validated GDF15 as a key driver of cancer cachexia and as a potential therapeutic target for the treatment and/or prevention of this syndrome.
RESUMEN
Dysregulation of Notch signaling has been implicated in the development of many different types of cancer. Notch inhibitors are being tested in the clinic, but in most cases gastrointestinal and other toxicities have limited the dosage and, therefore, the effectiveness of these therapies. Herein, we describe the generation of a monoclonal antibody against the ligand-binding domain of the Notch1 receptor that specifically blocks ligand-induced activation. This antibody, 23814, recognizes both human and murine Notch1 with similar affinity, enabling examination of the effects on both tumor and host tissue in preclinical models. 23814 blocked Notch1 function in vivo, inhibited functional angiogenesis, and inhibited tumor growth without causing gastrointestinal toxicity. The lack of toxicity allowed for combination of 23814 and the VEGFR inhibitor tivozanib, resulting in significant growth inhibition of several VEGFR inhibitor-resistant tumor models. Analysis of the gene expression profiles of an extensive collection of murine breast tumors enabled the successful prediction of which tumors were most likely to respond to the combination of 23814 and tivozanib. Therefore, the use of a specific Notch1 antibody that does not induce significant toxicity may allow combination treatment with angiogenesis inhibitors or other targeted agents to achieve enhanced therapeutic benefit.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neovascularización Patológica/metabolismo , Receptor Notch1/agonistas , Inhibidores de la Angiogénesis/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Anticuerpos Bloqueadores/toxicidad , Anticuerpos Monoclonales/toxicidad , Línea Celular Tumoral , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Femenino , Humanos , Ligandos , Ratones , Compuestos de Fenilurea/farmacología , Quinolinas/farmacología , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptores de Factores de Crecimiento Endotelial Vascular/metabolismo , Transducción de Señal/efectos de los fármacos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. EXPERIMENTAL DESIGN: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. RESULTS: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. CONCLUSIONS: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Expresión Génica , Neoplasias/genética , Neurregulina-1/genética , Receptor ErbB-3/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Área Bajo la Curva , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ligandos , Ratones , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Neurregulina-1/metabolismo , Pronóstico , Unión Proteica , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dysregulated fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We surveyed a broad panel of human cancer cell lines for the dysregulation of FGFR2 signaling and discovered that breast and gastric cancer cell lines harboring FGFR2 amplification predominantly express the IIIb isoform of the receptor. Therefore, we used an FGFR2-IIIb-specific antibody, GP369, to investigate the importance of FGFR2 signaling in vitro and in vivo. GP369 specifically and potently suppressed ligand-induced phosphorylation of FGFR2-IIIb and downstream signaling, as well as FGFR2-driven proliferation in vitro. The administration of GP369 in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support the hypothesis that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias/terapia , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/inmunología , Fosforilación/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tumor cells extensively utilize the pentose phosphate pathway for the synthesis of ribose. Transketolase is a key enzyme in this pathway and has been suggested as a target for inhibition in the treatment of cancer. In a pharmacodynamic study, nude mice with xenografted HCT-116 tumors were dosed with 1 ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of thiamine, the co-factor of transketolase. Transketolase activity was almost completely suppressed in blood, spleen, and tumor cells, but there was little effect on the activity of the other thiamine-utilizing enzymes alpha-ketoglutarate dehydrogenase or glucose-6-phosphate dehydrogenase. Synthesis and SAR of transketolase inhibitors is described.
Asunto(s)
Neoplasias del Colon/tratamiento farmacológico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Tiamina/análogos & derivados , Tiamina/antagonistas & inhibidores , Transcetolasa/antagonistas & inhibidores , Animales , Neoplasias del Colon/enzimología , Cristalografía por Rayos X , Glucosafosfato Deshidrogenasa/metabolismo , Humanos , Técnicas In Vitro , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Espectroscopía de Resonancia Magnética , Ratones , Ratones Desnudos , Estructura Molecular , Oxitiamina/antagonistas & inhibidores , Fosforilación/efectos de los fármacos , Bazo/efectos de los fármacos , Bazo/enzimología , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Inhibition of the thiamine-utilizing enzyme transketolase (TK) has been linked with diminished tumor cell proliferation. Most thiamine antagonists have a permanent positive charge on the B-ring, and it has been suggested that this charge is required for diphosphorylation by thiamine pyrophosphokinase (TPPK) and binding to TK. We sought to make neutral thiazolium replacements that would be substrates for TPPK, while not necessarily needing thiamine transporters (ThTr1 and ThTr2) for cell penetration. The synthesis, SAR, and structure-based rationale for highly potent non-thiazolium TK antagonists are presented.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Tiamina/análogos & derivados , Transcetolasa/antagonistas & inhibidores , Animales , Catálisis , Línea Celular , Cristalografía por Rayos X , Inhibidores Enzimáticos/química , Humanos , Ratones , Conformación Proteica , Relación Estructura-Actividad , Tiamina/química , Tiamina/farmacologíaRESUMEN
Transketolase, a key enzyme in the pentose phosphate pathway, has been suggested as a target for inhibition in the treatment of cancer. Compound 5a ('N3'-pyridyl thiamine'; 3-(6-methyl-2-amino-pyridin-3-ylmethyl)-5-(2-hydroxy-ethyl)-4-methyl-thiazol-3-ium chloride hydrochloride), an analog of the transketolase cofactor thiamine, is a potent transketolase inhibitor but suffers from poor pharmacokinetics due to high clearance and C(max) linked toxicity. An efficient way of improving the pharmacokinetic profile of 5a is to prepare oxidized prodrugs which are slowly reduced in vivo yielding longer, sustained blood levels of the drug. The synthesis of such prodrugs and their evaluation in rodent models is reported.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Profármacos/farmacología , Tiamina/análogos & derivados , Transcetolasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Estructura Molecular , Profármacos/química , Profármacos/farmacocinética , Tiamina/química , Tiamina/farmacocinética , Tiamina/farmacologíaRESUMEN
Saposin C binds to membranes to activate lipid degradation in lysosomes. To get insights into saposin C's function, we have determined its three-dimensional structure by NMR and investigated its interaction with phospholipid vesicles. Saposin C adopts the saposin-fold common to other members of the family. In contrast, the electrostatic surface revealed by the NMR structure is remarkably different. We suggest that charge distribution in the protein surface can modulate membrane interaction leading to the functional diversity of this family. We find that the binding of saposin C to phospholipid vesicles is a pH-controlled reversible process. The pH dependence of this interaction is sigmoidal, with an apparent pK(a) for binding close to 5.3. The pK(a) values of many solvent-exposed Glu residues are anomalously high and close to the binding pK(a). Our NMR data are consistent with the absence of a conformational change prior to membrane binding. All this information suggests that the negatively charged electrostatic surface of saposin C needs to be partially neutralized to trigger membrane binding. We have studied the membrane-binding behavior of a mutant of saposin C designed to decrease the negative charge of the electrostatic surface. The results support our conclusion on the importance of protein surface neutralization in binding. Since saposin C is a lysosomal protein and pH gradients occur in lysosomes, we propose that lipid degradation in the lysosome could be switched on and off by saposin C's reversible binding to membranes.
Asunto(s)
Glicoproteínas/química , Fosfolípidos/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Humanos , Concentración de Iones de Hidrógeno , Liposomas , Micelas , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Octoxinol , Fosfatidilcolinas/química , Fosfatidilserinas/química , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Saposinas , Soluciones , Relación Estructura-ActividadRESUMEN
The "BH3-only" proteins of the BCL-2 family require "multidomain" proapoptotic members BAX and BAK to release cytochrome c from mitochondria and kill cells. We find short peptides representing the alpha-helical BH3 domains of BID or BIM are capable of inducing oligomerization of BAK and BAX to release cytochrome c. Another subset characterized by the BH3 peptides from BAD and BIK cannot directly activate BAX, BAK but instead binds antiapoptotic BCL-2, resulting in the displacement of BID-like BH3 domains that initiate mitochondrial dysfunction. Transduced BAD-like and BID-like BH3 peptides also displayed synergy in killing leukemic cells. These data support a two-class model for BH3 domains: BID-like domains that "activate" BAX, BAK and BAD-like domains that "sensitize" by occupying the pocket of antiapoptotic members.
Asunto(s)
Apoptosis/fisiología , Mitocondrias/patología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Proteína Proapoptótica que Interacciona Mediante Dominios BH3 , Proteína 11 Similar a Bcl2 , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Grupo Citocromo c/metabolismo , Humanos , Células Jurkat , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Ratones , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/química , Proteínas Proto-Oncogénicas c-bcl-2/genética , Transducción Genética , Proteína Destructora del Antagonista Homólogo bcl-2 , Proteína X Asociada a bcl-2RESUMEN
The mechanism during apoptosis by which cytochrome c is rapidly and completely released in the absence of mitochondrial swelling is uncertain. Here, we show that two distinct pathways are involved. One mediates release of cytochrome c across the outer mitochondrial membrane, and another, characterized in this study, is responsible for the redistribution of cytochrome c stored in intramitochondrial cristae. We have found that the "BH3-only" molecule tBID induces a striking remodeling of mitochondrial structure with mobilization of the cytochrome c stores (approximately 85%) in cristae. This reorganization does not require tBID's BH3 domain and is independent of BAK, but is inhibited by CsA. During this process, individual cristae become fused and the junctions between the cristae and the intermembrane space are opened.