RESUMEN
Major limitations of camptothecin anticancer drugs (toxicity, nonselectivity, water insolubility, inactivation by human serum albumin) may be improved by creating glucuronide prodrugs that rely on beta-glucuronidase for their activation. We found that the camptothecin derivative 5,6-dihydro-4H-benzo[de]quinoline-camptothecin (BQC) displays greater cytotoxicity against cancer cells than the clinically used camptothecin derivatives SN-38 and topotecan even in the presence of human serum albumin. We synthesized the prodrug BQC-glucuronide (BQC-G), which was 4000 times more water soluble and 20-40 times less cytotoxic than BQC. Importantly, even in the presence of human serum albumin, BQC-G was efficiently hydrolyzed by beta-glucuronidase and produced greater cytotoxicity (IC50 = 13 nM) than camptothecin, 9-aminocamptothecin, SN-38, or topotecan (IC50 > 3000, 1370, 48, and 28 nM, respectively). BQC-G treatment of mice bearing human colon cancer xenografts with naturally or artificially elevated beta-glucuronidase activity produced significant antitumor activity, showing that BQC-G is a potent prodrug suitable for selective intratumoral drug activation.
Asunto(s)
Glucurónidos/química , Glucurónidos/farmacología , Profármacos/química , Profármacos/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Camptotecina/análogos & derivados , Camptotecina/química , Camptotecina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Neoplasias del Colon/tratamiento farmacológico , Femenino , Glucuronidasa/metabolismo , Glucurónidos/uso terapéutico , Humanos , Irinotecán , Ratones , Ratones Endogámicos BALB C , Profármacos/metabolismo , Profármacos/uso terapéutico , Topotecan/química , Topotecan/farmacologíaRESUMEN
CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/ßG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/ßG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/ßG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/ßG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.
Asunto(s)
Antineoplásicos/farmacología , Camptotecina/análogos & derivados , Escherichia coli/genética , Ingeniería Genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Animales , Antineoplásicos/metabolismo , Camptotecina/metabolismo , Camptotecina/farmacología , Línea Celular Tumoral , Escherichia coli/enzimología , Colorantes Fluorescentes/metabolismo , Terapia Genética , Humanos , Hidrólisis , Irinotecán , Ratones , Transformación GenéticaRESUMEN
Beta-glucuronidase (ßG) is a potential biomarker for cancer diagnosis and prodrug therapy. The ability to image ßG activity in patients would assist in personalized glucuronide prodrug cancer therapy. However, whole-body imaging of ßG activity for medical usage is not yet available. Here, we developed a radioactive ßG activity-based trapping probe for positron emission tomography (PET). We generated a (124)I-tyramine-conjugated difluoromethylphenol beta-glucuronide probe (TrapG) to form (124)I-TrapG that could be selectively activated by ßG for subsequent attachment of (124)I-tyramine to nucleophilic moieties near ßG-expressing sites. We estimated the specificity of a fluorescent FITC-TrapG, the cytotoxicity of tyramine-TrapG, and the serum half-life of (124)I-TrapG. ßG targeting of (124)I-TrapG in vivo was examined by micro-PET. The biodistribution of (131)I-TrapG was investigated in different organs. Finally, we imaged the endogenous ßG activity and assessed its correlation with therapeutic efficacy of 9-aminocamptothecin glucuronide (9ACG) prodrug in native tumors. FITC-TrapG showed specific trapping at ßG-expressing CT26 (CT26/mßG) cells but not in CT26 cells. The native TrapG probe possessed low cytotoxicity. (124)I-TrapG preferentially accumulated in CT26/mßG but not CT26 cells. Meanwhile, micro-PET and whole-body autoradiography results demonstrated that (124)I-TrapG signals in CT26/mßG tumors were 141.4-fold greater than in CT26 tumors. Importantly, Colo205 xenografts in nude mice that express elevated endogenous ßG can be monitored by using infrared glucuronide trapping probes (NIR-TrapG) and suppressed by 9ACG prodrug treatment. (124)I-TrapG exhibited low cytotoxicity allowing long-term monitoring of ßG activity in vivo to aid in the optimization of prodrug targeted therapy.
Asunto(s)
Glucuronidasa/metabolismo , Glucurónidos/uso terapéutico , Radioisótopos de Yodo , Tomografía de Emisión de Positrones , Profármacos , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Neoplasias/diagnóstico , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Tomografía de Emisión de Positrones/métodos , Sensibilidad y Especificidad , Distribución Tisular , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Membrane-tethered proteins (mammalian surface display) are increasingly being used for novel therapeutic and biotechnology applications. Maximizing surface expression of chimeric proteins on mammalian cells is important for these applications. We show that the cytoplasmic domain from the B7-1 antigen, a commonly used element for mammalian surface display, can enhance the intracellular transport and surface display of chimeric proteins in a Sar1 and Rab1 dependent fashion. However, mutational, alanine scanning and deletion analysis demonstrate the absence of linear ER export motifs in the B7 cytoplasmic domain. Rather, efficient intracellular transport correlated with the presence of predicted secondary structure in the cytoplasmic tail. Examination of the cytoplasmic domains of 984 human and 782 mouse type I transmembrane proteins revealed that many previously identified ER export motifs are rarely found in the cytoplasmic tail of type I transmembrane proteins. Our results suggest that efficient intracellular transport of B7 chimeric proteins is associated with the structure rather than to the presence of a linear ER export motif in the cytoplasmic tail, and indicate that short (less than ~ 10-20 amino acids) and unstructured cytoplasmic tails should be avoided to express high levels of chimeric proteins on mammalian cells.
Asunto(s)
Antígeno B7-1/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Western Blotting , Células Cultivadas , Citometría de Flujo , Glicosilación , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Transporte de Proteínas , Homología de Secuencia de Aminoácido , Anticuerpos de Cadena Única/metabolismoRESUMEN
Conventional cancer chemotherapy is limited by systemic toxicity and poor selectivity. Tumor-selective activation of glucuronide prodrugs by beta-glucuronidase in the tumor microenvironment in a monotherapeutic approach is one promising way to increase cancer selectivity. Here we examined the cellular requirement for enzymatic activation as well as the in vivo toxicity and antitumor activity of a glucuronide prodrug of a potent duocarmycin analogue that is active at low picomolar concentrations. Prodrug activation by intracellular and extracellular beta-glucuronidase was investigated by measuring prodrug 2 cytotoxicity against human cancer cell lines that displayed different endogenous levels of beta-glucuronidase, as well as against beta-glucuronidase-deficient fibroblasts and newly established beta-glucuronidase knockdown cancer lines. In all cases, glucuronide prodrug 2 was 1000-5000 times less cytotoxic than the parent duocarmycin analogue regardless of intracellular levels of beta-glucuronidase. By contrast, cancer cells that displayed tethered beta-glucuronidase on their plasma membrane were 80-fold more sensitive to glucuronide prodrug 2, demonstrating that prodrug activation depended primarily on extracellular rather than intracellular beta-glucuronidase activity. Glucuronide prodrug 2 (2.5 mg/kg) displayed greater antitumor activity and less systemic toxicity in vivo than the clinically used drug carboplatin (50 mg/kg) to mice bearing human lung cancer xenografts. Intratumoral injection of an adenoviral vector expressing membrane-tethered beta-glucuronidase dramatically enhanced the in vivo antitumor activity of prodrug 2. Our data provide evidence that increasing extracellular beta-glucuronidase activity in the tumor microenvironment can boost the therapeutic index of a highly potent glucuronide prodrug.
Asunto(s)
Antineoplásicos/metabolismo , Indoles/metabolismo , Profármacos/metabolismo , Adenoviridae/genética , Animales , Antineoplásicos/química , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Duocarmicinas , Líquido Extracelular/enzimología , Femenino , Vectores Genéticos , Glucuronidasa/antagonistas & inhibidores , Glucuronidasa/genética , Glucuronidasa/metabolismo , Glucurónidos/química , Glucurónidos/metabolismo , Glucurónidos/uso terapéutico , Humanos , Indoles/química , Indoles/uso terapéutico , Ratones , Ratones Desnudos , Estructura Molecular , Profármacos/química , Profármacos/uso terapéutico , Pirroles/química , Pirroles/metabolismo , Pirroles/uso terapéutico , ARN Interferente Pequeño/genética , Microambiente Tumoral , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
We describe an adjustable membrane-tethered/soluble protein screening methodology termed ECSTASY (enzyme cleavable surface tethered all-purpose screening system) which combines the power of high-throughput fluorescence-activated cell sorting of membrane-tethered proteins with the flexibility of soluble assays for isolation of improved mammalian recombinant proteins. In this approach, retroviral transduction is employed to stably tether a library of protein variants on the surface of mammalian cells via a glycosyl phosphatidylinositol anchor. High-throughput fluorescence-activated cell sorting is used to array cells expressing properly folded and/or active protein variants on their surface into microtiter culture plates. After culture to expand individual clones, treatment of cells with phosphatidylinositol-phospholipase C releases soluble protein variants for multiplex measurement of protein concentration, activity and/or function. We utilized ECSTASY to rapidly generate human ß-glucuronidase variants for cancer therapy by antibody-directed enzyme prodrug therapy with up to 30-fold greater potency to catalyze the hydrolysis of the clinically relevant camptothecin anti-cancer prodrug as compared with wild-type human ß-glucuronidase. A variety of recombinant proteins could be adjustably displayed on fibroblasts, suggesting that ECSTASY represents a general, simple and versatile methodology for high-throughput screening to accelerate sequence activity-based evolution of mammalian proteins.
Asunto(s)
Proteínas Ligadas a GPI/genética , Glucuronidasa/genética , Glucuronidasa/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Células 3T3 , Animales , Línea Celular , Proteínas Ligadas a GPI/metabolismo , Expresión Génica , Humanos , Ratones , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SolubilidadRESUMEN
Campylobacter jejuni is a common cause of pediatric diarrhea worldwide. Cytolethal distending toxin, produced by Campylobacter jejuni, is a putative virulence factor that induces cell cycle arrest and apoptosis in eukaryotic cells. Cellular cholesterol, a major component of lipid rafts, has a pivotal role in regulating signaling transduction and protein trafficking as well as pathogen internalization. In this study, we demonstrated that cell intoxication by Campylobacter jejuni cytolethal distending toxin is through the association of cytolethal distending toxin subunits and membrane cholesterol-rich microdomains. Cytolethal distending toxin subunits cofractionated with detergent-resistant membranes, while the distribution reduced upon the depletion of cholesterol, suggesting that cytolethal distending toxin subunits are associated with lipid rafts. The disruption of cholesterol using methyl-ß-cyclodextrin not only reduced the binding activity of cytolethal distending toxin subunits on the cell membrane but also impaired their delivery and attenuated toxin-induced cell cycle arrest. Accordingly, cell intoxication by cytolethal distending toxin was restored by cholesterol replenishment. These findings suggest that membrane cholesterol plays a critical role in the Campylobacter jejuni cytolethal distending toxin-induced pathogenesis of host cells.
Asunto(s)
Toxinas Bacterianas/metabolismo , Campylobacter jejuni/patogenicidad , Colesterol/metabolismo , Microdominios de Membrana/metabolismo , Animales , Toxinas Bacterianas/genética , Células CHO , Campylobacter jejuni/metabolismo , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Membrana Celular/metabolismo , Colesterol/farmacología , Cricetinae , Cricetulus , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Factores de Virulencia/metabolismo , beta-Ciclodextrinas/farmacologíaRESUMEN
Antibody-directed enzyme prodrug therapy (ADEPT) utilizing ß-glucuronidase is a promising method to enhance the therapeutic index of cancer chemotherapy. In this approach, an immunoenzyme (antibody-ß-glucuronidase fusion protein) is employed to selectively activate anticancer glucuronide prodrugs in the tumor microenvironment. A major roadblock to the clinical translation of this therapeutic strategy, however, is the low enzymatic activity and strong immunogenicity of the current generation of immunoenzymes. To overcome this problem, we fused a humanized single-chain antibody (scFv) of mAb CC49 to S2, a human ß-glucuronidase (hßG) variant that displays enhanced catalytic activity for prodrug hydrolysis. Here, we show that hcc49-S2 displayed 100-fold greater binding avidity than hcc49 scFv, possessed greater enzymatic activity than wild-type hßG, and more effectively killed antigen-positive cancer cells exposed to an anticancer glucuronide prodrug as compared to an analogous hßG immunoenzyme. Treatment of tumor-bearing mice with hcc49-S2 followed by prodrug significantly delayed tumor growth as compared to hcc49-hßG. Our study shows that hcc49-S2 is a promising targeted enzyme for cancer treatment and demonstrates that enhancement of human enzyme catalytic activity is a powerful approach to improve immunoenzyme efficacy.
Asunto(s)
Anticuerpos Antineoplásicos/metabolismo , Glucuronidasa/metabolismo , Glucurónidos/metabolismo , Profármacos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Microambiente Tumoral , Animales , Anticuerpos Antineoplásicos/química , Anticuerpos Antineoplásicos/aislamiento & purificación , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Biocatálisis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Glucuronidasa/química , Glucuronidasa/aislamiento & purificación , Glucurónidos/farmacología , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Ratones SCID , Imagen Molecular , Células 3T3 NIH , Profármacos/farmacología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Relación Estructura-Actividad , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: This study is aimed at investigating the in vivo antitumor activity of a novel cell-impermeable glucuronide prodrug, 9-aminocamptothecin glucuronide (9ACG), and elucidating the synergistically antitumor effects of antiangiogenesis therapy by targeting the tumor microenvironment. EXPERIMENTAL DESIGN: We analyzed the antitumor effects of 9ACG alone or combined with antiangiogenic monoclonal antibody DC101 on human tumor xenografts by measuring tumor growth and mouse survival in BALB/c nu/nu nude and NOD/SCID mice. The drug delivery, immune response, and angiogenesis status in treated tumors were assessed by high performance liquid chromatography, immunohistochemistry, and immunofluorescence assays. RESULTS: We developed a nontoxic and cell-impermeable glucuronide prodrug, 9ACG, which can only be activated by extracellular beta-glucuronidase to become severely toxic. 9ACG possesses potent antitumor activity against human tumor xenografts in BALB/c nu/nu nude mice but not for tumors implanted in NOD/SCID mice deficient in macrophages and neutrophils, suggesting that these cells play an important role in activating 9ACG in the tumor microenvironment. Most importantly, antiangiogenic monoclonal antibody DC101 potentiated single-dose 9ACG antitumor activity and prolonged survival of mice bearing resistant human colon tumor xenografts by providing strong beta-glucuronidase activity and prodrug delivery through enhancing inflammatory cell infiltration and normalizing tumor vessels in the tumor microenvironment. We also show that inflammatory cells (neutrophils) were highly infiltrated in advanced human colon cancer tissues compared with normal counterparts. CONCLUSIONS: Our study provides in vivo evidence that 9ACG has potential for prodrug monotherapy or in combination with antiangiognesis treatment for tumors with infiltration of macrophage or neutrophil inflammatory cells.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Neovascularización Patológica/prevención & control , Ensayos Antitumor por Modelo de Xenoinjerto , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacología , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Camptotecina/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Femenino , Glucurónidos/administración & dosificación , Glucurónidos/farmacología , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos NOD , Ratones Desnudos , Ratones SCID , Neoplasias/irrigación sanguínea , Neoplasias/patología , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Profármacos/administración & dosificación , Profármacos/farmacología , Carga Tumoral/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunologíaRESUMEN
Irinotecan (CPT-11) is a clinically important anticancer prodrug that requires enzymatic hydrolysis by carboxyesterase to generate the active metabolite SN-38. However, SN-38 is further metabolized to inactive SN-38 glucuronide (SN-38G), thus diminishing the levels of active SN-38. Although exogenously administered glucuronide drugs are being investigated for cancer therapy, it is unknown if endogenously generated camptothecin glucuronide metabolites can be used for tumor therapy. Here, we tested the hypothesis that tumor-located hydrolysis of endogenously generated SN-38G can enhance the antitumor efficacy of CPT-11 therapy. EJ human bladder carcinoma cells expressing membrane-tethered beta-glucuronidase (EJ/mbetaG cells) were used to selectively hydrolyze SN-38G to SN-38. Parental EJ and EJ/mbetaG cells displayed similar in vitro and in vivo growth rates and sensitivities to CPT-11 and SN-38. By contrast, EJ/mbetaG cells were more than 30 times more sensitive than EJ cells to SN-38G, showing that SN-38 could be generated from SN-38G in vitro. Systemic administration of CPT-11 resulted in tumor-located hydrolysis of SN-38G and accumulation of SN-38 in EJ/mbetaG subcutaneous tumors. Importantly, systemic administration of CPT-11, which itself is not a substrate for beta-glucuronidase, dramatically delayed the growth of EJ/mbetaG xenografts without increased systemic toxicity. Thus, the anticancer activity of CPT-11 can be significantly enhanced by converting the relatively high levels of endogenously generated SN-38G to SN-38 in tumors. The high concentrations of SN-38G found in the serum of patients treated with CPT-11 suggest that clinical response to CPT-11 may be improved by elevating beta-glucuronidase activity in tumors.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Camptotecina/análogos & derivados , Carboxilesterasa/metabolismo , Glucuronatos/farmacología , Glucuronidasa/metabolismo , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Camptotecina/farmacología , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Humanos , Irinotecán , Ratones , Ratones SCID , Inhibidores de Topoisomerasa I , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patologíaRESUMEN
Human beta-glucuronidase, due to low intrinsic immunogenicity in humans, is an attractive enzyme for tumor-specific prodrug activation, but its utility is hindered by low activity at physiological pH. Here we describe the development of a high-throughput screening procedure for enzymatic activity based on the stable retention of fluorescent reaction product in mammalian cells expressing properly folded glycoproteins on their surface. We utilized this procedure on error-prone PCR and saturation mutagenesis libraries to isolate beta-glucuronidase tetramers that were up to 60-fold more active (k(cat)/K(m)) at pH 7.0 and were up to an order of magnitude more effective at catalyzing the conversion of two structurally disparate glucuronide prodrugs to anticancer agents. The screening procedure described here can facilitate investigation of eukaryotic enzymes requiring posttranslational modifications for biological activity.
Asunto(s)
Glucuronidasa/genética , Lisosomas/enzimología , Proteínas de la Membrana/genética , Ingeniería de Proteínas , Animales , Variación Genética , Glucuronidasa/metabolismo , Glucuronidasa/farmacología , Humanos , Concentración de Iones de Hidrógeno , Lisosomas/genética , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Estructura Molecular , Profármacos/metabolismo , Regulación hacia ArribaRESUMEN
Expression of recombinant proteins in mammalian cells is useful for obtaining products with normal post-translational modifications. We describe a simple and economical method for the production of milligram levels of proteins in murine fibroblasts. Retroviral or LIPOFECTAMINE (Gibco Laboratories) transduction was employed to generate stable murine-fibroblast producer cells. Confluent cultures of stable fibroblast clones were maintained for up to 1 month in 0.5% serum. Culture medium was collected every 2-3 days and polyhistidine-tagged proteins were purified by ammonium sulphate precipitation and Ni(2+)-nitrilotriacetic acid affinity chromatography. Highly pure, active, glycosylated recombinant proteins, including human beta-glucuronidase, mouse beta-glucuronidase, aminopeptidase N (CD13) and a single-chain antibody-enzyme fusion protein, were obtained with yields of 3-6 mg/l of culture medium. Fc-tagged proteins were also produced and purified in a single step by Protein A affinity chromatography with yields of 6-12 mg/l. The techniques described here allow simple and economical production of recombinant mammalian proteins with post-translational modifications.