RESUMEN
BACKGROUND: Ovarian cancer is initially responsive to frontline chemotherapy. Unfortunately, it often recurs and becomes resistant to available therapies and the survival rate for advanced and recurrent ovarian cancer is unacceptably low. We thus hypothesized that it would be possible to achieve more durable treatment responses by combining cisplatin chemotherapy with SW IV-134, a cancer-targeted peptide mimetic and inducer of cell death. SW IV-134 is a recently developed small molecule conjugate linking a sigma-2 ligand with a peptide analog (mimetic) of the intrinsic death pathway activator SMAC (second-mitochondria activator of caspases). The sigma-2 receptor is overexpressed in ovarian cancer and the sigma-2 ligand portion of the conjugate facilitates cancer selectivity. The effector portion of the conjugate is expected to synergize with cisplatin chemotherapy and the cancer selectivity is expected to reduce putative off-target toxicities. METHODS: Ovarian cancer cell lines were treated with cisplatin alone, SW IV-134 alone and a combination of the two drugs. Treatment efficacy was determined using luminescent cell viability assays. Caspase-3/7, - 8 and - 9 activities were measured as complementary indicators of death pathway activation. Syngeneic mouse models and patient-derived xenograft (PDX) models of human ovarian cancer were studied for response to SW IV-134 and cisplatin monotherapy as well as combination therapy. Efficacy of the therapy was measured by tumor growth rate and survival as the primary readouts. Potential drug related toxicities were assessed at necropsy. RESULTS: The combination treatment was consistently superior in multiple cell lines when compared to the single agents in vitro. The expected mechanism of tumor cell death, such as caspase activation, was confirmed using luminescent and flow cytometry-based assay systems. Combination therapy proved to be superior in both syngeneic and PDX-based murine models of ovarian cancer. Most notably, combination therapy resulted in a complete resolution of established tumors in all study animals in a patient-derived xenograft model of ovarian cancer. CONCLUSIONS: The addition of SW IV-134 in combination with cisplatin chemotherapy represents a promising treatment option that warrants further pre-clinical development and evaluation as a therapy for women with advanced ovarian cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Compuestos de Azabiciclo/uso terapéutico , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Cisplatino/uso terapéutico , Oligopéptidos/uso terapéutico , Neoplasias Ováricas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Línea Celular Tumoral , Resistencia a Antineoplásicos , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Despite considerable efforts by scientific research, pancreatic cancer is the fourth leading cause of cancer related mortalities. Sigma-2 receptors, which are overexpressed in several tumors, represent promising targets for triggering selective pancreatic cancer cells death. METHODS: We selected five differently structured high-affinity sigma-2 ligands (PB28, PB183, PB221, F281 and PB282) to study how they affect the viability of diverse pancreatic cancer cells (human cell lines BxPC3, AsPC1, Mia PaCa-2, and Panc1 and mouse Panc-02, KCKO and KP-02) and how this is reflected in vivo in a tumor model. RESULTS: Important cytotoxicity was shown by the compounds in the aggressive Panc02 cells, where cytotoxic activity was caspase-3 independent for four of the five compounds. However, both cytotoxicity and caspase-3 activation involved generation of Reactive Oxygen Species (ROS), which could be partially reverted by the lipid antioxidant α-tocopherol, but not by the hydrophilic N-acetylcysteine (NAC) indicating crucial differences in the intracellular sites exposed to oxidative stress induced by sigma-2 receptor ligands. Importantly, all the compounds strongly increased the production of mitochondrial superoxide radicals except for PB282. Despite a poor match between in vitro and the in vivo efficacy, daily treatment of C57BL/6 mice bearing Panc02 tumors resulted in promising effects with PB28 and PB282 which were similar compared to the current standard-of-care chemotherapeutic gemcitabine without showing signs of systemic toxicities. CONCLUSIONS: Overall, this study identified differential sensitivities of pancreatic cancer cells to structurally diverse sigma-2 receptor ligands. Of note, we identified the mitochondrial superoxide pathway as a previously unrecognized sigma-2 receptor-activated process, which encourages further studies on sigma-2 ligand-mediated cancer cell death for the targeted treatment of pancreatic tumors.
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Caspasa 3/metabolismo , Muerte Celular/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neoplasias Pancreáticas/tratamiento farmacológico , Piperazinas/farmacología , Receptores sigma/agonistas , Superóxidos/metabolismo , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Femenino , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Neoplasias Pancreáticas/metabolismo , Piperazina , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Autosomal dominant retinal vasculopathy with cerebral leukodystrophy is a microvascular endotheliopathy with middle-age onset. In nine families, we identified heterozygous C-terminal frameshift mutations in TREX1, which encodes a 3'-5' exonuclease. These truncated proteins retain exonuclease activity but lose normal perinuclear localization. These data have implications for the maintenance of vascular integrity in the degenerative cerebral microangiopathies leading to stroke and dementias.
Asunto(s)
Encefalopatías/genética , Exodesoxirribonucleasas/genética , Mutación , Fosfoproteínas/genética , Enfermedades de la Retina/genética , Secuencia de Aminoácidos , Encefalopatías/enzimología , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Genes Dominantes , Predisposición Genética a la Enfermedad , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Microscopía Confocal , Datos de Secuencia Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Enfermedades de la Retina/enzimología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
BACKGROUND: Drug resistance is a significant problem in the treatment of ovarian cancer and can be caused by multiple mechanisms. Inhibition of apoptosis by the inhibitor of apoptosis proteins (IAPs) represents one such mechanism, and can be overcome by a mitochondrial protein called second mitochondria-derived activator of caspases (SMAC). We have previously shown that the ligands of sigma-2 receptors effectively induce tumor cell death. Additionally, because sigma-2 receptors are preferentially expressed in tumor cells, their ligands provide an effective mechanism for selective anti-cancer therapy. METHODS: In the current work, we have improved upon the previously described sigma-2 ligand SW43 by conjugating it to a pro-apoptotic small molecule SMAC mimetic SW IV-52, thus generating the novel cancer therapeutic SW IV-134. The new cancer drug was tested for receptor selectivity and tumor cell killing activity in vitro and in vivo. RESULTS: We have shown that SW IV-134 retained adequate sigma-2 receptor binding affinity in the context of the conjugate and potently induced cell death in ovarian cancer cells. The cell death induced by SW IV-134 was significantly greater than that observed with either SW43 or SW IV-52 alone and in combination. Furthermore, the intraperitoneal administration of SW IV-134 significantly reduced tumor burden and improved overall survival in a mouse xenograft model of ovarian cancer without causing significant adverse effects to normal tissues. Mechanistically, SW IV-134 induced degradation of cIAP-1 and cIAP-2 leading to NF-ÒB activation and TNFα-dependent cell death. CONCLUSIONS: Our findings suggest that coupling sigma-2 ligands to SMAC peptidomimetics enhances their effectiveness while maintaining the cancer selectivity. This encouraging proof-of-principle preclinical study supports further development of tumor-targeted small peptide mimetics via ligands to the sigma-2 receptor for future clinical applications.
Asunto(s)
Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Compuestos de Azabiciclo/química , Compuestos de Azabiciclo/farmacología , Oligopéptidos/química , Oligopéptidos/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Receptores sigma/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis , Materiales Biomiméticos/química , Materiales Biomiméticos/farmacología , Western Blotting , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Femenino , Xenoinjertos , Humanos , Péptidos y Proteínas de Señalización Intracelular/química , Ligandos , Ratones , Ratones SCID , Proteínas Mitocondriales/química , Terapia Molecular Dirigida/métodos , Neoplasias Ováricas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Thrombin generates fibrin and activates platelets and endothelium, causing thrombosis and inflammation. Endothelial thrombomodulin (TM) changes thrombin's substrate specificity toward cleavage of plasma protein C into activated protein C (APC), which opposes its thrombotic and inflammatory activities. Endogenous TM activity is suppressed in pathologic conditions, and antithrombotic interventions involving soluble TM are limited by rapid blood clearance. To overcome this problem, we fused TM with a single chain fragment (scFv) of an antibody targeted to red blood cells. scFv/TM catalyzes thrombin-mediated generation of activated protein C and binds to circulating RBCs without apparent damage, thereby prolonging its circulation time and bioavailability orders of magnitude compared with soluble TM. In animal models, a single dose of scFv/TM, but not soluble TM, prevents platelet activation and vascular occlusion by clots. Thus, scFv/TM serves as a prodrug and provides thromboprophylaxis at low doses (0.15 mg/kg) via multifaceted mechanisms inhibiting platelets and coagulation.
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Quimioprevención/métodos , Sistemas de Liberación de Medicamentos/métodos , Eritrocitos/efectos de los fármacos , Trombomodulina/administración & dosificación , Trombosis/prevención & control , Animales , Células Cultivadas , Drosophila , Eritrocitos/metabolismo , Eritrocitos/fisiología , Humanos , Ratones , Modelos Biológicos , Terapia Molecular Dirigida/métodos , Unión Proteica , Proteína C/metabolismo , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/uso terapéutico , Anticuerpos de Cadena Única/administración & dosificación , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/metabolismo , Anticuerpos de Cadena Única/uso terapéutico , Trombomodulina/química , Trombomodulina/metabolismo , Trombomodulina/uso terapéuticoRESUMEN
BACKGROUND: The targeted delivery of cancer therapeutics represents an ongoing challenge in the field of drug development. TRAIL is a promising cancer drug but its activity profile could benefit from a cancer-selective delivery mechanism, which would reduce potential side effects and increase treatment efficiencies. We recently developed the novel TRAIL-based drug platform TR3, a genetically fused trimer with the capacity for further molecular modifications such as the addition of tumor-directed targeting moieties. MUC16 (CA125) is a well characterized biomarker in several human malignancies including ovarian, pancreatic and breast cancer. Mesothelin is known to interact with MUC16 with high affinity. In order to deliver TR3 selectively to MUC16-expressing cancers, we investigated the possibility of targeted TR3 delivery employing the high affinity mesothelin/MUC16 ligand/receptor interaction. METHODS: Using genetic engineering, we designed the novel cancer drug Meso-TR3, a fusion protein between native mesothelin and TR3. The recombinant proteins were produced with mammalian HEK293T cells. Meso-TR3 was characterized for binding selectivity and killing efficacy against MUC16-positive cancer cells and controls that lack MUC16 expression. Drug efficacy experiments were performed in vitro and in vivo employing an intraperitoneal xenograft mouse model of ovarian cancer. RESULTS: Similar to soluble mesothelin itself, the strong MUC16 binding property was retained in the Meso-TR3 fusion protein. The high affinity ligand/receptor interaction was associated with a selective accumulation of the cancer drug on MUC16-expressing cancer targets and directly correlated with increased killing activity in vitro and in a xenograft mouse model of ovarian cancer. The relevance of the mesothelin/MUC16 interaction for attaching Meso-TR3 to the cancer cells was verified by competitive blocking experiments using soluble mesothelin. Mechanistic studies using soluble DR5-Fc and caspase blocking assays confirmed engagement of the extrinsic death receptor pathway. Compared to non-targeted TR3, Meso-TR3 displayed a much reduced killing potency on cells that lack MUC16. CONCLUSIONS: Soluble Meso-TR3 targets the cancer biomarker MUC16 in vitro and in vivo. Following attachment to the tumor via surface bound MUC16, Meso-TR3 acquires full activation with superior killing profiles compared to non-targeted TR3, while its bioactivity is substantially reduced on cells that lack the tumor marker. This prodrug phenomenon represents a highly desirable property because it has the potential to enhance cancer killing with fewer side-effects than non-targeted TRAIL-based therapeutics. Thus, further exploration of this novel fusion protein is warranted as a possible therapeutic for patients with MUC16-positive malignancies.
Asunto(s)
Antineoplásicos/farmacología , Antígeno Ca-125/metabolismo , Portadores de Fármacos , Proteínas Ligadas a GPI/metabolismo , Proteínas de la Membrana/metabolismo , Neoplasias Ováricas/tratamiento farmacológico , Ligando Inductor de Apoptosis Relacionado con TNF/farmacología , Animales , Antineoplásicos/metabolismo , Relación Dosis-Respuesta a Droga , Femenino , Células HEK293 , Células HeLa , Humanos , Células Jurkat , Ligandos , Mesotelina , Ratones , Ratones Endogámicos NOD , Ratones SCID , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Unión Proteica , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismo , Factores de Tiempo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer.
Asunto(s)
Antineoplásicos , Neoplasias Ováricas , Humanos , Animales , Ratones , Femenino , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Antineoplásicos/química , Apoptosis , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Proteínas Inhibidoras de la Apoptosis/metabolismo , Caspasas/metabolismo , Línea Celular TumoralRESUMEN
Sigma-2-ligands (S2L) are characterized by high binding affinities to their cognate sigma-2 receptor, overexpressed in rapidly proliferating tumor cells. As such, S2L were developed as imaging probes (ISO1) or as cancer therapeutics, alone (SV119 [C6], SW43 [C10]) and as delivery vehicles for cytotoxic drug cargoes (C6-Erastin, C10-SMAC). However, the exact mechanism of S2L-induced cytotoxicity remains to be fully elucidated. A series of high-affinity S2L were evaluated regarding their cytotoxicity profiles across cancer cell lines. While C6 and C10 displayed distinct cytotoxicities, C0 and ISO1 were essentially non-toxic. Confocal microscopy and lipidomics analysis in cellular and mouse models revealed that C10 induced increases in intralysosomal free cholesterol and in cholesterol esters, suggestive of unaltered intracellular cholesterol trafficking. Cytotoxicity was caused by cholesterol excess, a phenomenon that contrasts the effects of NPC1 inhibition. RNA-sequencing revealed gene clusters involved in cholesterol homeostasis and ER stress response exclusively by cytotoxic S2L. ER stress markers were confirmed by qPCR and their targeted modulation inhibited or enhanced cytotoxicity of C10 in a predicted manner. Moreover, C10 increased sterol regulatory element-binding protein 2 (SREBP2) and low-density lipoprotein receptor (LDLR), both found to be pro-survival factors activated by ER stress. Furthermore, inhibition of downstream processes of the adaptive response to S2L with simvastatin resulted in synergistic treatment outcomes in combination with C10. Of note, the S2L conjugates retained the ER stress response of the parental ligands, indicative of cholesterol homeostasis being involved in the overall cytotoxicity of the drug conjugates. Based on these findings, we conclude that S2L-mediated cell death is due to free cholesterol accumulation that leads to ER stress. Consequently, the cytotoxic profiles of S2L drug conjugates are proposed to be enhanced via concurrent ER stress inducers or simvastatin, strategies that could be instrumental on the path toward tumor eradication.
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Colesterol , Estrés del Retículo Endoplásmico , Receptores sigma , Colesterol/metabolismo , Receptores sigma/metabolismo , Receptores sigma/genética , Humanos , Animales , Ratones , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ligandos , Línea Celular Tumoral , Muerte Celular/efectos de los fármacos , Neoplasias/metabolismo , Neoplasias/tratamiento farmacológico , Neoplasias/patologíaRESUMEN
NK-cell killing requires both the expression of activating receptor ligands and low MHC class I expression by target cells. Here we demonstrate that the expression of any of the murine ligands for the NK-cell activating receptor NKG2D results in a concomitant reduction in MHC class I expression. We show this both in tumor cell lines and in vivo. NK-cell lysis is enhanced by the decrease in MHC class I expression, suggesting the change is biologically relevant. These results demonstrate that NKG2D ligand expression on target cells not only allows for activating receptor recognition, but also actively reduces expression of the inhibitory ligand, MHC class I, leading to enhanced recognition and killing by NK cells.
Asunto(s)
Células Asesinas Naturales/inmunología , Subfamilia K de Receptores Similares a Lectina de Células NK/inmunología , Animales , Línea Celular Tumoral , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase I/inmunología , Antígenos de Histocompatibilidad Clase I/metabolismo , Células Asesinas Naturales/metabolismo , Ligandos , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Subfamilia K de Receptores Similares a Lectina de Células NK/genética , Subfamilia K de Receptores Similares a Lectina de Células NK/metabolismo , Proteínas Asociadas a Matriz Nuclear/genética , Proteínas Asociadas a Matriz Nuclear/inmunología , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/inmunologíaRESUMEN
Plasminogen activators (PAs) are used to treat life-threatening thrombosis, but not for thromboprophylaxis because of rapid clearance, risk of bleeding, and central nervous system (CNS) toxicity. We describe a novel strategy that may help to overcome these limitations by targeting a thrombin-activated PA pro-drug to circulating red blood cells (RBCs). We fused a single chain antibody (scFv Ter-119) that binds to mouse glycophorin A (GPA) with a variant human single-chain low molecular weight urokinase construct that can be activated selectively by thrombin (scFv/uPA-T). scFv/uPA-T bound specifically to mouse RBCs without altering their biocompatibility and retained its zymogenic properties until converted by thrombin into an active 2-chain molecule. As a result, RBC-bound scFv/uPA-T caused thrombin-induced fibrinolysis. One hour and 48 hours after intravenous (IV) injection in mice, approximately 70% and approximately 35% of scFv/uPA-T was retained in the blood, respectively, and approximately 95% of the circulating scFv/uPA-T remained bound to RBCs. A single IV injection of scFv/uPA-T provided effective prophylaxis against arterial and venous thrombosis for up to 24 hours. Thus, prophylactic delivery of RBC-targeted PA pro-drugs activated selectively at the site of clot formation represents a new approach to prevent thrombosis in clinical settings where the risk of clotting is high.
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Sistemas de Liberación de Medicamentos , Precursores Enzimáticos/farmacología , Eritrocitos , Fibrinolíticos/farmacología , Profármacos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Trombosis/prevención & control , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Animales , Humanos , Ratones , Proteínas Recombinantes/farmacología , Factores de TiempoRESUMEN
PURPOSE: To investigate the metabolism of synovial sarcoma (SS) and elucidate the effect of malic enzyme 1 absence on SS redox homeostasis. EXPERIMENTAL DESIGN: ME1 expression was measured in SS clinical samples, SS cell lines, and tumors from an SS mouse model. The effect of ME1 absence on glucose metabolism was evaluated utilizing Seahorse assays, metabolomics, and C13 tracings. The impact of ME1 absence on SS redox homeostasis was evaluated by metabolomics, cell death assays with inhibitors of antioxidant systems, and measurements of intracellular reactive oxygen species (ROS). The susceptibility of ME1-null SS to ferroptosis induction was interrogated in vitro and in vivo. RESULTS: ME1 absence in SS was confirmed in clinical samples, SS cell lines, and an SS tumor model. Investigation of SS glucose metabolism revealed that ME1-null cells exhibit higher rates of glycolysis and higher flux of glucose into the pentose phosphate pathway (PPP), which is necessary to produce NADPH. Evaluation of cellular redox homeostasis demonstrated that ME1 absence shifts dependence from the glutathione system to the thioredoxin system. Concomitantly, ME1 absence drives the accumulation of ROS and labile iron. ROS and iron accumulation enhances the susceptibility of ME1-null cells to ferroptosis induction with inhibitors of xCT (erastin and ACXT-3102). In vivo xenograft models of ME1-null SS demonstrate significantly increased tumor response to ACXT-3102 compared with ME1-expressing controls. CONCLUSIONS: These findings demonstrate the translational potential of targeting redox homeostasis in ME1-null cancers and establish the preclinical rationale for a phase I trial of ACXT-3102 in SS patients. See related commentary by Subbiah and Gan, p. 3408.
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Ferroptosis , Sarcoma Sinovial , Animales , Antioxidantes , Ferroptosis/genética , Glucosa/metabolismo , Humanos , Hierro , Malato Deshidrogenasa , Ratones , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Glycoprotein CD44 and alternative splice variants are overexpressed in many cancers and cancer stem cells. Binding of hyaluronic acid to CD44 activates cell signaling pathways, inducing cell proliferation, cell survival, and invasion. As such, CD44 is regarded as an excellent target for cancer therapy when this interaction can be blocked. In this study, we developed a CD44-specific antibody fragment and evaluated it for imaging CD44-positive cancers using PET. Methods: A human single-chain fragment variable (scFv) was generated by phage display, using the extracellular domain of recombinant human CD44. The specificity and affinity of the scFv-CD44 were evaluated using recombinant and tumor cell-expressed CD44. Epitope mapping of the putative CD44 binding site was performed via overlapping peptide microarray. The scFv-CD44 was reformatted into a bivalent scFv-Fc-CD44, based on human IgG1-fragment crystallizable (Fc). The scFv-Fc-CD44 was radiolabeled with 64Cu and 89Zr. The purified reagents were injected into athymic nude mice bearing CD44-positive human tumors (MDA-MB-231, breast cancer, triple-negative). Biodistribution studies were performed at different times after injection of [64Cu]Cu-NOTA-scFv-Fc-CD44 or [89Zr]Zr-DFO-scFv-Fc-CD44. PET/CT imaging was conducted with [89Zr]Zr-DFO-scFv-Fc-CD44 on days 1 and 7 after injection and compared with a scFv-Fc control antibody construct targeting glycophorin A. Results: Epitope mapping of the scFv binding site revealed a linear epitope within the extracellular domain of human CD44, capable of blocking binding to native hyaluronic acid. Switching from a monovalent scFv to a bivalent scFv-Fc format improved its binding affinity toward native CD44 on human breast cancer cells by nearly 200-fold. In vivo biodistribution data showed the highest tumor uptake and tumor-to-blood ratios for [89Zr]Zr-DFO-scFv-Fc-CD44 between days 5 and 7. PET imaging confirmed excellent tumor specificity for [89Zr]Zr-DFO-scFv-Fc-CD44 when compared with the control scFv-Fc. Conclusion: We developed a CD44-specific scFv-Fc construct that binds with nanomolar affinity to human CD44. When radiolabeled with 64Cu or 89Zr, it demonstrated specific uptake in CD44-expressing MDA-MB-231 tumors. The high tumor uptake (â¼56% injected dose/g) warrants clinical investigation of [89Zr]Zr-DFO-scFv-Fc-CD44 as a versatile PET imaging agent for patients with CD44-positive tumors.
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Receptores de Hialuranos/metabolismo , Ingeniería de Proteínas , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Animales , Línea Celular Tumoral , Cristalización , Humanos , Ratones , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/farmacocinética , Distribución TisularRESUMEN
BACKGROUND: Sigma-2 receptors are over-expressed in proliferating cancer cells, making an attractive target for the targeted treatment of pancreatic cancer. In this study, we investigated the role of the novel sigma-2 receptor ligand SW43 to induce apoptosis and augment standard chemotherapy. RESULTS: The binding affinity for sigma-2 ligands is high in pancreas cancer, and they induce apoptosis with a rank order of SV119 < SW43 < SRM in vitro. Combining these compounds with gemcitabine further increased apoptosis and decreased viability. Our in vivo model showed that sigma-2 ligand treatment decreased tumor volume to the same extent as gemcitabine. However, SW43 combination treatment with gemcitabine was superior to the other compounds and resulted in stabilization of tumor volume during treatment, with minimal toxicities. CONCLUSIONS: This study shows that the sigma-2 ligand SW43 has the greatest capacity to augment gemcitabine in a pre-clinical model of pancreas cancer and has provided us with the rationale to move this compound forward with clinical investigations for patients with pancreatic cancer.
Asunto(s)
Antineoplásicos/uso terapéutico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores sigma/metabolismo , Animales , Antineoplásicos/farmacocinética , Caspasa 3/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/farmacología , Desoxicitidina/uso terapéutico , Femenino , Citometría de Flujo , Humanos , Ratones , Ratones Endogámicos C57BL , Receptores sigma/agonistas , GemcitabinaRESUMEN
Chemical coupling to carrier red blood cells (RBCs) converts tissue type plasminogen activator (tPA) from a problematic therapeutic into a safe agent for thromboprophylaxis. The goal of this study was to develop a more clinically relevant recombinant biotherapeutic by fusing a mutant tPA with a single-chain antibody fragment (scFv) with specificity for glycophorin A (GPA) on mouse RBCs. The fusion construct (anti-GPA scFv/PA) bound specifically to mouse but not human RBCs and activated plasminogen; this led to rapid and stable attachment of up to 30,000 copies of anti-GPA scFv/PA per mouse RBC that were thereby endowed with high fibrinolytic activity. Binding of anti-GPA scFv/PA neither caused RBC aggregation, hemolysis, uptake in capillary-rich lungs or in the reticuloendothelial system nor otherwise altered the circulation of RBCs. Over 40% of labeled anti-GPA scFv/PA injected in mice bound to RBC, which markedly prolonged its intravascular circulation and fibrinolytic activity compared with its nontargeted PA counterpart, anti-GPA scFv/PA, but not its nontargeted PA analog, prevented thrombotic occlusion in FeCl(3) models of vascular injury. These results provide proof-of-principle for the development of a recombinant PA variant that binds to circulating RBC and provides thromboprophylaxis by use of a clinically relevant approach.
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Eritrocitos/efectos de los fármacos , Fibrinolíticos/farmacología , Activadores Plasminogénicos/farmacología , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/farmacología , Animales , Agregación Eritrocitaria , Eritrocitos/fisiología , Fibrinolíticos/farmacocinética , Glicoforinas/inmunología , Hemólisis , Humanos , Técnicas In Vitro , Venas Yugulares , Ratones , Ratones Endogámicos C57BL , Mutación , Activadores Plasminogénicos/genética , Activadores Plasminogénicos/farmacocinética , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacocinética , Anticuerpos de Cadena Única/genética , Trombosis de la Vena/sangre , Trombosis de la Vena/prevención & controlRESUMEN
Complement activation is tightly regulated to avoid excessive inflammatory and immune responses. Crry(-/-) is an embryonic lethal phenotype secondary to the maternal complement alternative pathway (AP) attacking a placenta deficient in this inhibitor. In this study, we demonstrate that Crry(-/-) mice could be rescued on a partial as well as on a complete factor B (fB)- or C3-deficient maternal background. The C3 and fB protein concentrations in Crry(-/-)C3(+/-) and Crry(-/-)fB(+/-) mice were substantially reduced for gene dosage secondary to enhanced AP turnover. Based on these observations, a breeding strategy featuring reduced maternal AP-activating capacity rescued the lethal phenotype. It led to a novel, stable line of Crry SKO mice carrying normal alleles for C3 and fB. Crry SKO mice also had accelerated C3 and fB turnover and therefore reduced AP- activating potential. These instructive results represent an example of a membrane regulatory protein being responsible for homeostasis of the complement system. They imply that there is constant turnover on cells of the AP pathway which functions as an immune surveillance system for pathogens and altered self.
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Vía Alternativa del Complemento/inmunología , Homeostasis/inmunología , Proteínas de la Membrana/fisiología , Receptores de Complemento/fisiología , Animales , Línea Celular , Complemento C3/biosíntesis , Complemento C3/deficiencia , Complemento C3/metabolismo , Factor B del Complemento/biosíntesis , Factor B del Complemento/deficiencia , Factor B del Complemento/genética , Vía Alternativa del Complemento/genética , Pérdida del Embrión/genética , Pérdida del Embrión/inmunología , Femenino , Genotipo , Homeostasis/genética , Humanos , Masculino , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de Complemento/deficiencia , Receptores de Complemento/genética , Receptores de Complemento 3bRESUMEN
The aggressiveness of pancreatic cancer urgently requires more efficient treatment options. Because the sigma-2 (σ2) receptor was recently proposed as a promising target for pancreatic cancer therapy, we explored our previously developed multifunctional thiosemicarbazones, designed to synergistically impair cell energy levels, by targeting σ2 and P-gp proteins and chelating Iron. A deconstruction approach was herein applied by removing one function at a time from the potent multifunctional thiosemicarbazones 1 and 2, to investigate the contribution to cytotoxicity of each target involved. The results from in vitro (panel of pancreatic tumor cells) and in vivo experiments (C57BL/6 bearing KP02 tumor), suggest that while the multifunctional activity was not required for the antitumor activity of these thiosemicarbazones, σ2-targeting appeared to allow alternative tumor cell death mechanisms, leading to potent and less toxic off-targets toxicities compared to other thiosemicarbazones devoid of σ2-targeting.
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Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/farmacología , Quelantes/farmacología , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores sigma/antagonistas & inhibidores , Tiosemicarbazonas/farmacología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Muerte Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Quelantes/síntesis química , Quelantes/química , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Femenino , Humanos , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Receptores sigma/metabolismo , Relación Estructura-Actividad , Tiosemicarbazonas/síntesis química , Tiosemicarbazonas/químicaRESUMEN
Clinical application of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-based cancer therapeutics has not reached optimal potencies in part due to inadequate drug stability and inefficiencies in cancer-selective drug delivery. As such, innovative strategies regarding drug design and delivery are of utmost importance to achieve improved treatment results. With our current study, we aimed at exploring the groundwork for a two-stage targeting concept, which is based on the intrinsic tumor homing capacity of mesenchymal stem cells (MSCs) as cellular drug factories for the in situ production of our newly designed and biomarker-targeted TRAIL-based TR3 therapeutics. Since MSCs are primary cells, capable in vitro of only a limited number of cell divisions, identification of suitable strategies for their efficient genetic manipulation is of critical importance. We chose adenoviral (Ad) vectors as a transduction vehicle due to its ability to infect dividing and non-dividing cells and because of their limited restrictions regarding the packaging capacity of their genetic payload. In order to enhance the transduction efficacy of MSCs using Ad5 wild-type-based vectors, we tested a variety of fiber knob modifications on a panel of patient-derived MSC lines established from adipose tissue. We identified Ad5pK7, an Ad5 vector containing a polylysine fiber knob modification, exhibiting the highest transduction rates across a panel of 16 patient-derived MSC lines. We further demonstrated that MSCs could be efficiently transduced with an Ad5pK7 vector containing membrane-anchored and secreted TR3 expression units, including the MUC16 (CA125)-targeted variant Meso64-TR3. In both in vitro and in vivo experiments, MSC-derived Meso64-TR3 was far more potent on MUC16-expressing ovarian cancer compared to its non-targeted TR3 counterpart. Our findings thus provide the foundation to initiate further preclinical investigations on MSC-mediated treatment options in ovarian cancer using biomarker-targeted TR3-based biologics.
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Adenoviridae/genética , Productos Biológicos/uso terapéutico , Células Madre Mesenquimatosas/citología , Neoplasias Ováricas/terapia , Ligando Inductor de Apoptosis Relacionado con TNF/uso terapéutico , Transducción Genética , Femenino , Humanos , Neoplasias Ováricas/patologíaRESUMEN
BACKGROUND: Pancreatic cancer is a lethal malignancy that frequently acquires resistance to conventional chemotherapies often associated with overexpression of inhibitors of apoptosis proteins (IAPs). We have recently described a novel means to deliver second mitochondria-derived activator of caspases (SMAC) mimetics selectively to cancer cells employing the sigma-2 ligand/receptor interaction. The intrinsic death pathway agonist SMAC offers an excellent opportunity to counteract the anti-apoptotic activity of IAPs. SMAC mimetics have been used to sensitize several cancer types to chemotherapeutic agents but cancer-selective delivery and appropriate cellular localization have not yet been considered. In our current study, we tested the ability of the sigma-2/SMAC drug conjugate SW IV-134 to sensitize pancreatic cancer cells to gemcitabine. METHODS: Using the targeted SMAC mimetic SW IV-134, inhibition of the X-linked inhibitor of apoptosis proteins (XIAP) was induced pharmacologically and its impact on cell viability was studied alone and in combination with gemcitabine. Pathway analyses were performed by assessing caspase activation, PARP cleavage and membrane blebbing (Annexin-V), key components of apoptotic cell death. Single-agent treatment regimens were compared with combination therapy in a preclinical mouse model of pancreatic cancer. RESULTS: The sensitizing effect of XIAP interference toward gemcitabine was confirmed via pharmacological intervention using our recently designed, targeted SMAC mimetic SW IV-134 across a wide range of commonly used pancreatic cancer cell lines at concentrations where the individual drugs showed only minimal activity. On a mechanistic level, we identified involvement of key components of the apoptosis machinery during cell death execution. Furthermore, combination therapy proved superior in decreasing the tumor burden and extending the lives of the animals in a preclinical mouse model of pancreatic cancer. CONCLUSION: We believe that the strong sensitizing capacity of SW IV-134 in combination with clinically relevant doses of gemcitabine represents a promising treatment option that warrants clinical evaluation.
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Antimetabolitos Antineoplásicos/administración & dosificación , Antineoplásicos/administración & dosificación , Compuestos de Azabiciclo/administración & dosificación , Desoxicitidina/análogos & derivados , Oligopéptidos/administración & dosificación , Neoplasias Pancreáticas/tratamiento farmacológico , Animales , Antimetabolitos Antineoplásicos/farmacología , Antineoplásicos/farmacología , Compuestos de Azabiciclo/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/farmacología , Sinergismo Farmacológico , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Oligopéptidos/farmacología , Neoplasias Pancreáticas/metabolismo , Proteína Inhibidora de la Apoptosis Ligada a X/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
TRAIL continues to garner substantial interest as a recombinant cancer therapeutic while the native cytokine itself serves important tumor surveillance functions when expressed in membrane-anchored form on activated immune effector cells. We have recently developed the genetically stabilized TRAIL platform TR3 in efforts to improve the limitations associated with currently available drug variants. While in the process of characterizing mesothelin-targeted TR3 variants using a single chain antibody (scFv) delivery format (SS-TR3), we discovered that the membrane-tethered cytokine had a substantially increased activity profile compared to non-targeted TR3. However, cell death proceeded exclusively via a bystander mechanism and protected the mesothelin-positive targets from apoptosis rather than leading to their elimination. Incorporation of a spacer-into the mesothelin surface antigen or the cancer drug itself-converted SS-TR3 into a cis-acting phenotype. Further experiments with membrane-anchored TR3 variants and the native cytokine confirmed our hypothesis that membrane-proximal TRAIL species lack the capacity to physically engage their cognate receptors coexpressed on the same cell membrane. Our findings not only provide an explanation for the "peaceful" coexistence of ligand and receptor of a representative member of the TNF superfamily but give us vital clues for the design of activity-enhanced TR3-based cancer therapeutics.
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Anticuerpos Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Neoplasias , Anticuerpos de Cadena Única/farmacología , Ligando Inductor de Apoptosis Relacionado con TNF , Animales , Células CHO , Cricetinae , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Ensayos de Selección de Medicamentos Antitumorales/métodos , Células HEK293 , Humanos , Células Jurkat , Mesotelina , Ratones , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Ligando Inductor de Apoptosis Relacionado con TNF/antagonistas & inhibidores , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
TRAIL has been extensively explored as a cancer drug based on its tumor-selective activity profile but it is incapable per se of discriminating between death receptors expressed by normal host cells and transformed cancer cells. Furthermore, it is well documented that surface tethering substantially increases its biologic activity. We have previously reported on Meso-TR3, a constitutive TRAIL trimer targeted to the biomarker MUC16 (CA125), in which the entire ectodomain of human mesothelin was genetically fused to the TR3 platform, facilitating attachment to the cancer cells via the MUC16 receptor. Here, we designed a truncation variant, in which the minimal 64 amino acid MUC16 binding domain of mesothelin was incorporated into TR3. It turned out that the dual-domain biologic Meso64-TR3 retained its high MUC16 affinity and bound to the cancer cells quickly, independent of the TR3/death receptor interaction. Furthermore, it was substantially more potent than Meso-TR3 and TR3 in vitro and in a preclinical xenograft model of MUC16-dependent ovarian cancer. Phenotypically, Meso64-TR3 is more closely related to non-targeted TR3, evident by indistinguishable activity profiles on MUC16-deficient cancers and similar thermal stability characteristics. Overall, Meso64-TR3 represents a fully human, MUC16-targetd TRAIL-based biologic, ideally suited for exploring preclinical and clinical evaluation studies in MUC16-dependent malignancies.