RESUMEN
Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated in situ and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA-protein fusion. HER2 affibody-mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody-mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.
RESUMEN
AIM: We endeavored to create a folate-targeted liposome (Fol-liposome) that could selectively target areas of inflammation. MATERIALS & METHODS: Fol-liposomes were prepared with encapsulated DiD fluorophore or betamethasone (BM) to image and treat an adjuvant-induced rat model of rheumatoid arthritis. RESULTS: Fol-liposomes selectively accumulated in arthritic rat paws to a greater extent than nontargeted liposomes. When these Fol-liposomes were used to encapsulate BM and administered to arthritic rats, animals exhibited less paw swelling, lower arthritis scores, a reduction in bone erosion, less splenomegaly and better maintenance of body weight when compared with nontreated or nontargeted BM-containing liposome groups. CONCLUSION: Fol-liposomes can selectively deliver imaging and therapeutic agents to sites of inflammation in a rat model of rheumatoid arthritis.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Ácido Fólico/química , Ácido Fólico/metabolismo , Liposomas/química , Terapia Molecular Dirigida/métodos , Adyuvantes Inmunológicos/metabolismo , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Liberación de Fármacos , Femenino , Colorantes Fluorescentes/química , Transportadores de Ácido Fólico/metabolismo , Inflamación/tratamiento farmacológico , Macrófagos , Imagen Óptica/métodos , Tamaño de la Partícula , Ratas , Ratas Endogámicas Lew , Propiedades de SuperficieRESUMEN
Radiolabeling of substrates with 2-[18F]fluoroethylazide exploits the rapid kinetics, chemical selectivity, and mild conditions of the copper-catalyzed azide-alkyne cycloaddition reaction. While this methodology has proven to result in near-quantitative labeling of alkyne-tagged precursors, the relatively small size of the fluoroethylazide group makes separation of the 18F-labeled radiotracer and the unreacted precursor challenging, particularly with precursors >500 Da (e.g., peptides). We have developed an inexpensive azide-functionalized resin to rapidly remove unreacted alkyne precursor following the fluoroethylazide labeling reaction and integrated it into a fully automated radiosynthesis platform. We have carried out 2-[18F]fluoroethylazide labeling of four different alkynes ranging from <300 Da to >1700 Da and found that >98% of the unreacted alkyne was removed in less than 20 min at room temperature to afford the final radiotracers at >99% radiochemical purity with specific activities up to >200 GBq/µmol. We have applied this technique to label a novel cyclic peptide previously evolved to bind the Her2 receptor with high affinity, and demonstrated tumor-specific uptake and low nonspecific background by PET/CT. This resin-based methodology is automated, rapid, mild, and general allowing peptide-based fluorine-18 radiotracers to be obtained with clinically relevant specific activities without chromatographic separation and with only a minimal increase in total synthesis time.
Asunto(s)
Alquinos/química , Azidas/química , Radioisótopos de Flúor/química , Péptidos Cíclicos/química , Tomografía Computarizada por Tomografía de Emisión de Positrones/métodos , Química Clic/métodos , Cobre/química , Reacción de Cicloadición/métodosRESUMEN
BACKGROUND: Autophagy is a bulk catabolic process that modulates tumorigenesis, therapeutic resistance, and dormancy. The tumor suppressor ARHI (DIRAS3) is a potent inducer of autophagy and its expression results in necroptotic cell death in vitro and tumor dormancy in vivo. ARHI is down-regulated or lost in over 60 % of primary ovarian tumors yet is dramatically up-regulated in metastatic disease. The metabolic changes that occur during ARHI induction and their role in modulating death and dormancy are unknown. METHODS: We employed Nuclear Magnetic Resonance (NMR)-based metabolomic strategies to characterize changes in key metabolic pathways in both cell culture and xenograft models of ARHI expression and autophagy. These pathways were further interrogated by cell-based immunofluorescence imaging, tracer uptake studies, targeted metabolic inhibition, and in vivo PET/CT imaging. RESULTS: Induction of ARHI in cell culture models resulted in an autophagy-dependent increase in lactate production along with increased glucose uptake and enhanced sensitivity to glycolytic inhibitors. Increased uptake of glutamine was also dependent on autophagy and dramatically sensitized cultured ARHI-expressing ovarian cancer cell lines to glutaminase inhibition. Induction of ARHI resulted in a reduction in mitochondrial respiration, decreased mitochondrial membrane potential, and decreased Tom20 staining suggesting an ARHI-dependent loss of mitochondrial function. ARHI induction in mouse xenograft models resulted in an increase in free amino acids, a transient increase in [18F]-FDG uptake, and significantly altered choline metabolism. CONCLUSIONS: ARHI expression has previously been shown to trigger autophagy-associated necroptosis in cell culture. In this study, we have demonstrated that ARHI expression results in decreased cellular ATP/ADP, increased oxidative stress, and decreased mitochondrial function. While this bioenergetic shock is consistent with programmed necrosis, our data indicates that the accompanying up-regulation of glycolysis and glutaminolysis is autophagy-dependent and serves to support cell viability rather than facilitate necroptotic cell death. While the mechanistic basis for metabolic up-regulation following ARHI induction is unknown, our preliminary data suggest that decreased mitochondrial function and increased metabolic demand may play a role. These alterations in fundamental metabolic pathways during autophagy-associated necroptosis may provide the basis for new therapeutic strategies for the treatment of dormant ovarian tumors.
Asunto(s)
Autofagia , Redes y Vías Metabólicas , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Proteínas de Unión al GTP rho/metabolismo , Animales , Línea Celular Tumoral , Modelos Animales de Enfermedad , Femenino , Glucosa/metabolismo , Glutamatos/metabolismo , Glutamina/metabolismo , Glucólisis , Xenoinjertos , Humanos , Potencial de la Membrana Mitocondrial , Ratones , Mitocondrias/metabolismo , Neoplasias Ováricas/diagnóstico por imagen , Estrés Oxidativo , Tomografía Computarizada por Tomografía de Emisión de PositronesRESUMEN
Peptides typically have poor biostabilities, and natural sequences cannot easily be converted into drug-like molecules without extensive medicinal chemistry. We have adapted mRNA display to drive the evolution of highly stable cyclic peptides while preserving target affinity. To do this, we incorporated an unnatural amino acid in an mRNA display library that was subjected to proteolysis prior to selection for function. The resulting "SUPR (scanning unnatural protease resistant) peptide" showed ≈500-fold improvement in serum stability (t1/2 =160â h) and up to 3700-fold improvement in protease resistance versus the parent sequence. We extended this approach by carrying out SUPR peptide selections against Her2-positive cells in culture. The resulting SUPR4 peptide showed low-nanomolar affinity toward Her2, excellent specificity, and selective tumor uptake in vivo. These results argue that this is a general method to design potent and stable peptides for in vivo imaging and therapy.
Asunto(s)
Evolución Molecular Dirigida , Péptido Hidrolasas/metabolismo , Péptidos Cíclicos/química , Péptidos Cíclicos/metabolismo , Aminoácidos/química , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Línea Celular Tumoral , Humanos , Ratones , Modelos Moleculares , Estructura Molecular , Biblioteca de Péptidos , Péptidos Cíclicos/farmacocinética , Estabilidad Proteica , ARN Mensajero/genética , Células Tumorales CultivadasRESUMEN
PURPOSE: Although current therapies for many inflammatory/autoimmune diseases are effective, a significant number of patients still exhibit only partial or negligible responses to therapeutic intervention. Since prolonged use of an inadequate therapy can result in both progressive tissue damage and unnecessary expense, methods to identify nonresponding patients are necessary. PROCEDURES: Four murine models of inflammatory disease (rheumatoid arthritis, ulcerative colitis, pulmonary fibrosis, and atherosclerosis) were induced, treated with anti-inflammatory agents, and evaluated for inflammatory response. The mice were also injected intraperitoneally with OTL0038, a folate receptor-targeted near-infrared dye that accumulates in activated macrophages at sites of inflammation. Uptake of OTL0038 in inflamed lesions was then correlated with clinical measurements of disease severity. RESULTS: OTL0038 accumulated at sites of inflammation in all four animal models. More importantly, changes in lesion-associated OTL0038 preceded changes in clinical symptoms in mice treated with all anti-inflammatory drugs examined. CONCLUSION: OTL0038 has the ability to predict responses to multiple therapies in four murine models of inflammation.
Asunto(s)
Enfermedades Autoinmunes/terapia , Receptores de Folato Anclados a GPI/metabolismo , Inflamación/terapia , Imagen Molecular/métodos , Animales , Artritis Reumatoide/complicaciones , Artritis Reumatoide/patología , Artritis Reumatoide/terapia , Aterosclerosis/complicaciones , Aterosclerosis/patología , Aterosclerosis/terapia , Enfermedades Autoinmunes/complicaciones , Enfermedades Autoinmunes/patología , Bovinos , Colitis Ulcerosa/complicaciones , Colitis Ulcerosa/patología , Colitis Ulcerosa/terapia , Colon/patología , Femenino , Fluorescencia , Inflamación/complicaciones , Inflamación/patología , Masculino , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Fibrosis Pulmonar/complicaciones , Fibrosis Pulmonar/patología , Fibrosis Pulmonar/terapia , Resultado del TratamientoRESUMEN
The ability to select patients who will respond to therapy is especially acute for autoimmune/inflammatory diseases, where the costs of therapies can be high and the progressive damage associated with ineffective treatments can be irreversible. In this article we describe a clinical test that will rapidly predict the response of patients with an autoimmune/inflammatory disease to many commonly employed therapies. This test involves quantitative assessment of uptake of a folate receptor-targeted radioimaging agent ((99m)Tc-EC20) by a subset of inflammatory macrophages that accumulate at sites of inflammation. Murine models of four representative inflammatory diseases (rheumatoid arthritis, inflammatory bowel disease, pulmonary fibrosis, and atherosclerosis) show markedly decreased uptake of (99m)Tc-EC20 in inflamed lesions upon initiation of successful therapies, but no decrease in uptake upon administration of ineffective therapies, in both cases long before changes in clinical symptoms can be detected. This predictive capability should reduce costs and minimize morbidities associated with failed autoimmune/inflammatory disease therapies.
Asunto(s)
Antiinflamatorios/uso terapéutico , Enfermedades Autoinmunes/tratamiento farmacológico , Factores Inmunológicos/uso terapéutico , Inflamación/tratamiento farmacológico , Macrófagos/efectos de los fármacos , Compuestos de Organotecnecio/farmacología , Animales , Antiinflamatorios/farmacología , Artritis Experimental/tratamiento farmacológico , Aterosclerosis/tratamiento farmacológico , Colitis Ulcerosa/tratamiento farmacológico , Modelos Animales de Enfermedad , Femenino , Citometría de Flujo , Receptores de Folato Anclados a GPI/efectos de los fármacos , Factores Inmunológicos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Fibrosis Pulmonar/tratamiento farmacológico , Resultado del TratamientoRESUMEN
Targeted therapies are emerging as a preferred strategy for the treatment of cancer and other diseases. To evaluate the impact of a high affinity targeting ligand on the rate and extent of tumor penetration of different sized nanomedicines, we have used intravital multiphoton microscopy to quantitate the kinetics of tumor accumulation of a homologous series of folate-PEG-rhodamine conjugates prepared with polyethylene glycols (PEG) of different molecular weights. We demonstrate that increasing the size of the folate-PEG-rhodamine conjugates results in both longer circulation times and slower tumor penetration rates. Although a "binding site barrier" is observed with the folate-linked polymers in folate receptor expressing tumors, ligand targeting eventually leads to increased tumor accumulation, with endocytosis of the targeted nanocarriers contributing to their enhanced tumor retention. Because the effects of nanocarrier size, shape, chemistry, and targeting ligand are interconnected and complex, we suggest that these parameters must be carefully optimized for each nanocarrier to ensure optimal drug delivery in vivo.
Asunto(s)
Ácido Fólico/farmacocinética , Nanopartículas , Neoplasias/metabolismo , Línea Celular Tumoral , Fluoresceína/química , Ácido Fólico/química , Humanos , Peso Molecular , Tamaño de la Partícula , Polietilenglicoles/químicaRESUMEN
Complete surgical resection of malignant disease is the only reliable method to cure cancer. Unfortunately, quantitative tumor resection is often limited by a surgeon's ability to locate all malignant disease and distinguish it from healthy tissue. Fluorescence-guided surgery has emerged as a tool to aid surgeons in the identification and removal of malignant lesions. While nontargeted fluorescent dyes have been shown to passively accumulate in some tumors, the resulting tumor-to-background ratios are often poor, and the boundaries between malignant and healthy tissues can be difficult to define. To circumvent these problems, our laboratory has developed high affinity tumor targeting ligands that bind to receptors that are overexpressed on cancer cells and deliver attached molecules selectively into these cells. In this study, we explore the use of two tumor-specific targeting ligands (i.e., folic acid that targets the folate receptor (FR) and DUPA that targets prostate specific membrane antigen (PSMA)) to deliver near-infrared (NIR) fluorescent dyes specifically to FR and PSMA expressing cancers, thereby rendering only the malignant cells highly fluorescent. We report here that all FR- and PSMA-targeted NIR probes examined bind cultured cancer cells in the low nanomolar range. Moreover, upon intravenous injection into tumor-bearing mice with metastatic disease, these same ligand-NIR dye conjugates render receptor-expressing tumor tissues fluorescent, enabling their facile resection with minimal contamination from healthy tissues.