RESUMEN
Altered energy metabolism is a cancer hallmark as malignant cells tailor their metabolic pathways to meet their energy requirements. Glucose and glutamine are the major nutrients that fuel cellular metabolism, and the pathways utilizing these nutrients are often altered in cancer. Here, we show that the long ncRNA CCAT2, located at the 8q24 amplicon on cancer risk-associated rs6983267 SNP, regulates cancer metabolism in vitro and in vivo in an allele-specific manner by binding the Cleavage Factor I (CFIm) complex with distinct affinities for the two subunits (CFIm25 and CFIm68). The CCAT2 interaction with the CFIm complex fine-tunes the alternative splicing of Glutaminase (GLS) by selecting the poly(A) site in intron 14 of the precursor mRNA. These findings uncover a complex, allele-specific regulatory mechanism of cancer metabolism orchestrated by the two alleles of a long ncRNA.
Asunto(s)
Glutaminasa/genética , Neoplasias/metabolismo , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Factores de Escisión y Poliadenilación de ARNm/metabolismo , Alelos , Empalme Alternativo , Metabolismo Energético , Células HCT116 , Humanos , Neoplasias/genética , Precursores del ARN/química , Precursores del ARN/metabolismo , ARN Mensajero/metabolismoRESUMEN
BACKGROUND: The fusion protein VEGF(121)/rGel composed of the growth factor VEGF(121) and the plant toxin gelonin targets the tumor neovasculature and exerts impressive anti-vascular effects. We have previously shown that VEGF(121)/rGel is cytotoxic to endothelial cells overexpressing VEGFR-2 but not to endothelial cells overexpressing VEGFR-1. In this study, we examined the basis for the specific toxicity of this construct and assessed its intracellular effects in vitro and in vivo. METHODS: We investigated the binding, cytotoxicity and internalization profile of VEGF(121)/rGel on endothelial cells expressing VEGFR-1 or VEGFR-2, identified its effects on angiogenesis models in vitro and ex vivo, and explored its intracellular effects on a number of molecular pathways using microarray analysis. RESULTS: Incubation of PAE/VEGFR-2 and PAE/VEGFR-1 cells with (125)I-VEGF(121)/rGel demonstrated binding specificity that was competed with unlabeled VEGF(121)/rGel but not with unlabeled gelonin. Assessment of the effect of VEGF(121)/rGel on blocking tube formation in vitro revealed a 100-fold difference in IC(50) levels between PAE/VEGFR-2 (1 nM) and PAE/VEGFR-1 (100 nM) cells. VEGF(121)/rGel entered PAE/VEGFR-2 cells within one hour of treatment but was not detected in PAE/VEGFR-1 cells up to 24 hours after treatment. In vascularization studies using chicken chorioallantoic membranes, 1 nM VEGF(121)/rGel completely inhibited bFGF-stimulated neovascular growth. The cytotoxic effects of VEGF(121)/rGel were not apoptotic since treated cells were TUNEL-negative with no evidence of PARP cleavage or alteration in the protein levels of select apoptotic markers. Microarray analysis of VEGF(121)/rGel-treated HUVECs revealed the upregulation of a unique "fingerprint" profile of 22 genes that control cell adhesion, apoptosis, transcription regulation, chemotaxis, and inflammatory response. CONCLUSIONS: Taken together, these data confirm the selectivity of VEGF(121)/rGel for VEGFR-2-overexpressing endothelial cells and represent the first analysis of genes governing intoxication of mammalian endothelial cells by a gelonin-based targeted therapeutic agent.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Células Endoteliales/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1/farmacología , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Aorta/citología , Embrión de Pollo , Pollos , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Sistemas de Liberación de Medicamentos , Células Endoteliales de la Vena Umbilical Humana , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Inactivadoras de Ribosomas Tipo 1/genética , Porcinos , Factor A de Crecimiento Endotelial Vascular/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Advances in recombinant DNA technology have opened up new possibilities of exploiting toxic proteins for therapeutic purposes. Bringing forth these protein toxins from the bench to the bedside strongly depends on the availability of production methods that are reproducible, scalable and comply with good manufacturing practice (GMP). The type I ribosome-inhibiting protein, gelonin, has great potential as an anticancer drug, but is sequestrated in endosomes and lysosomes. This can be overcome by combination with photochemical internalization (PCI), a method for endosomal drug release. The combination of gelonin-based drugs and PCI represents a tumor-targeted therapy with high precision and efficiency. The aim of this study was to produce recombinant gelonin (rGel) at high purity and quantity using an automated liquid chromatography system. The expression and purification process was documented as highly efficient (4.4 mg gelonin per litre induced culture) and reproducible with minimal loss of target protein (~50% overall yield compared to after initial immobilized metal affinity chromatography (IMAC)). The endotoxin level of 0.05-0.09 EU/mg was compatible with current standards for parenteral drug administration. The automated system provided a consistent output with minimal human intervention and close monitoring of each purification step enabled optimization of both yield and purity of the product. rGel was shown to have equivalent biological activity and cytotoxicity, both with and without PCI-mediated delivery, as rGelref produced without an automated system. This study presents a highly refined and automated manufacturing procedure for recombinant gelonin at a quantity and quality sufficient for preclinical evaluation. The methods established in this report are in compliance with high quality standards and compose a solid platform for preclinical development of gelonin-based drugs.
Asunto(s)
Cromatografía Liquida/métodos , Proteínas Inactivadoras de Ribosomas Tipo 1/biosíntesis , Antineoplásicos Fitogénicos/biosíntesis , Automatización , Línea Celular , Humanos , Proteínas de Plantas/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Toxinas Biológicas/biosíntesisRESUMEN
A commonly employed method to determine the function of a particular cell population and to assess its contribution to the overall system in vivo is to selectively deplete that population and observe the effects. Using monoclonal antibodies to deliver toxins to target cells can achieve this with a high degree of efficiency. Here, we describe an in vivo model combining the use of immunotoxins and multidrug resistant (MDR) gene deficient mice so that only MDR deficient cells expressing the target molecule would be depleted while target molecule expressing, but MDR sufficient, cells are spared. This allows targeted depletion at a higher degree of specificity than has been previously achieved. We have applied this technique to study trogocytosis, the intercellular transfer of cell surface molecules, but this principle could also be adapted using technology already available for use in other fields of study.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Citotoxicidad Inmunológica/efectos de los fármacos , Genes MDR/fisiología , Inmunotoxinas/toxicidad , Depleción Linfocítica/métodos , Subfamilia B de Transportador de Casetes de Unión a ATP/deficiencia , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/deficiencia , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Animales , Femenino , Supervivencia de Injerto/efectos de los fármacos , Trasplante de Corazón , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunoconjugados/toxicidad , Fragmentos Fab de Inmunoglobulinas/toxicidad , Trasplante de Riñón , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Noqueados , Proteínas Inactivadoras de Ribosomas Tipo 1/toxicidad , Bazo/efectos de los fármacos , Bazo/inmunología , Bazo/patología , Tolerancia al Trasplante/efectos de los fármacosRESUMEN
BACKGROUND: Antibody-drug conjugates are an exceptional and useful therapeutic tool for multiple diseases, particularly for cancer treatment. We previously showed that the fusion of the serine protease granzyme B (GrB), the effector molecule or T and B cells, to a binding domain allows the controlled and effective delivery of the cytotoxic payload into the target cell. The production of these constructs induced the formation of high molecular aggregates with a potential impact on the efficacy and safety of the protein. METHODS: Our laboratory designed a new Fn14 targeted fusion construct designated GrB(C210A)-Fc-IT4 which contains a modified GrB payload for improved protein production and preserved biological activity. We assessed the construct's enzymatic activity, as well as in vitro cytotoxicity and internalization into target cells. We also assessed pharmacokinetics, efficacy and toxicology parameters in vivo. RESULTS: GrB(C210A)-Fc-IT4 protein exhibited high affinity and selective cytotoxicity within the nanomolar range when tested against a panel of Fn14-positive human cancer cell lines. The construct rapidly internalized into target cells, activating the caspase cascade and causing mitochondrial membrane depolarization. Pharmacokinetic studies in mice revealed that GrB(C210A)-Fc-IT4 displayed a bi-exponential clearance from plasma with a fast initial clearance (t1/2α=0.36 hour) followed by a prolonged terminal-phase plasma half-life (t1/2ß=35 hours). Mice bearing MDA-MB-231 orthotopic tumor xenografts treated with vehicle or GrB(C210A)-Fc-IT4 construct (QODx5) demonstrated tumor regression and long-term (>80 days) suppression of tumor growth. Treatment of mice bearing established, subcutaneous A549 lung tumors showed impressive, long-term tumor suppression compared with a control group treated with vehicle alone. Administration of GrB(C210A)-Fc-IT4 (100 mg/kg total dose) was well-tolerated by mice and resulted in significant reduction of tumor burden in a lung cancer patient-derived xenograft model. Toxicity studies revealed no statistically significant changes in aspartate transferase, alanine transferase or lactate dehydrogenase in treated mice. Histopathological analysis of tissues from treated mice did not demonstrate any specific drug-related changes. CONCLUSION: GrB(C210A)-Fc-IT4 demonstrated excellent, specific cytotoxicity in vitro and impressive in vivo efficacy with no significant toxicity in normal murine models. These studies show GrB(C210A)-Fc-IT4 is an excellent candidate for further preclinical development.
Asunto(s)
Sistemas de Liberación de Medicamentos/métodos , Granzimas/metabolismo , Receptor de TWEAK/metabolismo , Animales , Femenino , Humanos , Ratones , Ratones DesnudosRESUMEN
Human single-chain Fv directed against fibroblast growth factor receptor 3 (FGFR3) have been shown to block proliferation of RT112 bladder carcinoma cells in vitro. Here, we examined the ability of the recombinant gelonin toxin (rGel) to enhance this inhibitory effect in vitro and in vivo on the bladder cancer cell line RT112 and the corresponding xenografts. Immunotoxins were genetically engineered by fusing FGFR3-specific Fv fragments (3C) to the NH(2) terminus of rGel and expressed as a soluble protein in Escherichia coli. The 3C/rGel fusion construct showed an IC(50) of 200 nmol/L against log-phase RT112 cells compared with 1,500 nmol/L for free rGel. Immunofluorescence studies showed that the 3C/rGel construct internalized rapidly into the cytoplasm of RT112 cells within 1 h of exposure. The mechanism of immunotoxin-induced cell death was found to be mediated by apoptosis. RT112 tumor xenografts in severe combined immunodeficient mice treated with 50 mg/kg 3C/rGel exhibited considerable growth delay relative to control tumors and a significant reduction of 55% to 70% in mean tumor size. Immunohistochemical analysis showed that tumors from mice treated with 3C/rGel displayed considerable apoptotic damage compared with control groups. Subcellular location of FGFR3 in immunotoxin-treated tumors indicated a translocation of FGFR3 to the nuclear membrane in contrast to tumors from saline-treated controls. These results show that FGFR3-driven immunotoxins may be an effective therapeutic agent against human bladder and other tumor types overexpressing FGFR3.
Asunto(s)
Antineoplásicos Fitogénicos/uso terapéutico , Apoptosis/efectos de los fármacos , Inmunotoxinas , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1/uso terapéutico , Neoplasias de la Vejiga Urinaria/tratamiento farmacológico , Animales , Carcinoma de Células Transicionales/tratamiento farmacológico , Carcinoma de Células Transicionales/metabolismo , Carcinoma de Células Transicionales/patología , Núcleo Celular/metabolismo , Proliferación Celular/efectos de los fármacos , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Fragmentos de Inmunoglobulinas , Ratones , Ratones Desnudos , Ratones SCID , Transporte de Proteínas , ARN Interferente Pequeño/farmacología , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 3 de Factor de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/uso terapéutico , Resonancia por Plasmón de Superficie , Tasa de Supervivencia , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/metabolismo , Neoplasias de la Vejiga Urinaria/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Immunotherapeutic approaches designed to augment T and B cell mediated killing of tumor cells has met with clinical success in recent years suggesting tremendous potential for treatment in a broad spectrum of tumor types. After complex recognition of target cells by T and B cells, delivery of the serine protease granzyme B (GrB) to tumor cells comprises the cytotoxic insult resulting in a well-characterized, multimodal apoptotic cascade. METHODS: We designed a recombinant fusion construct, GrB-Fc-4D5, composed of a humanized anti-HER2 scFv fused to active GrB for recognition of tumor cells and internal delivery of GrB, simulating T and B cell therapy. We assessed the construct's antigen-binding specificity and GrB enzymatic activity, as well as in vitro cytotoxicity and internalization into target and control cells. We also assessed pharmacokinetic and toxicology parameters in vivo. RESULTS: GrB-Fc-4D5 was highly cytotoxic to Her2 positive cells such as SKBR3, MCF7 and MDA-MB-231 with IC50 values of 56, 99 and 27 nM, respectively, and against a panel of HER2+ cell lines regardless of endogenous expression levels of the PI-9 inhibitor. Contemporaneous studies with Kadcyla demonstrated similar levels of in vitro activity against virtually all cells tested. GrB-Fc-4D5 internalized rapidly into target SKOV3 cells within 1 h of exposure rapidly delivering GrB to the cytoplasmic compartment. In keeping with its relatively high molecular weight (160 kDa), the construct demonstrated a terminal-phase serum half-life in mice of 39.2 h. Toxicity studies conducted on BALB/c mice demonstrated no statistically significant changes in SGPT, SGOT or serum LDH. Histopathologic analysis of tissues from treated mice demonstrated no drug-related changes in any tissues examined. CONCLUSION: GrB-Fc-4D5 shows excellent, specific cytotoxicity and demonstrates no significant toxicity in normal, antigen-negative murine models. This construct constitutes a novel approach against HER2-expressing tumors and is an excellent candidate for further development.
Asunto(s)
Antineoplásicos Inmunológicos/farmacología , Desarrollo de Medicamentos , Terapia Molecular Dirigida , Receptor ErbB-2/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/farmacología , Animales , Antígenos de Neoplasias/inmunología , Antineoplásicos Inmunológicos/aislamiento & purificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Sistemas de Liberación de Medicamentos , Expresión Génica , Vectores Genéticos/genética , Granzimas/administración & dosificación , Granzimas/genética , Humanos , Ratones , Terapia Molecular Dirigida/métodos , Unión Proteica/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Anticuerpos de Cadena Única/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The objective of this study was to develop and explore a novel CD133-targeting immunotoxin (IT) for use in combination with the endosomal escape method photochemical internalization (PCI). scFvCD133/rGelonin was recombinantly constructed by fusing a gene (scFvCD133) encoding the scFv that targets both non-glycosylated and glycosylated forms of both human and murine CD133/prominin-1 to a gene encoding the ribosome-inactivating protein (RIP) gelonin (rGelonin). RIP-activity was assessed in a cell-free translation assay. Selective binding and intracellular accumulation of scFvCD133/rGelonin was evaluated by flow cytometry and fluorescence microscopy. PCI of scFvCD133/rGelonin was explored in CD133high and CD133low cell lines and a CD133neg cell line, where cytotoxicity was evaluated by the MTT assay. scFvCD133/rGelonin exhibited superior binding to and a higher accumulation in CD133high cells compared to CD133low cells. No cytotoxic responses were detected in either CD133high or CD133low cells after 72 h incubation with <100 nM scFvCD133/rGelonin. Despite a severe loss in RIP-activity of scFvCD133/rGelonin compared to free rGelonin, PCI of scFvCD133/rGelonin induced log-fold reduction of viability compared to PCI of rGelonin. Strikingly, PCI of scFvCD133/rGelonin exceeded the cytotoxicity of PCI of rGelonin also in CD133low cells. In conclusion, PCI promotes strong cytotoxic activity of the per se non-toxic scFvCD133/rGelonin in both CD133high and CD133low cancer cells.
RESUMEN
B lymphocyte stimulator (BLyS) is crucial for B-cell survival, and the biological effects of BLyS are mediated by three cell surface receptors designated B cell-activating factor receptor (BAFF-R), transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), and B-cell maturation antibody (BCMA). Increased expression of BLyS and its receptors has been identified in numerous B-cell malignancies. We generated a fusion toxin designated rGel/BLyS for receptor-mediated delivery of the recombinant gelonin (rGel) toxin to neoplastic B cells, and we characterized its activity against various B-cell tumor lines. Three mantle cell lymphoma (MCL) cell lines (JeKo-1, Mino, and SP53) and two diffuse large B-cell lymphoma (DLBCL) cell lines (SUDHL-6 and OCI-Ly3) expressing all three distinct BLyS receptors were found to be the most sensitive to the fusion toxin (IC(50) = 2-5 pmol/L and 0.001-5 nmol/L for MCL and DLBCL, respectively). The rGel/BLyS fusion toxin showed specific binding to cells expressing BLyS receptors and rapid internalization of the rGel component into target cells. The cytotoxic effects of rGel/BLyS were inhibited by pretreatment with free BLyS or with soluble BAFF-R, TACI, and BCMA decoy receptors. This suggests that the cytotoxic effects of the fusion toxin are mediated through BLyS receptors. The rGel/BLyS fusion toxin inhibited MCL cell growth through induction of apoptosis associated with caspase-3 activation and poly (ADP-ribose) polymerase cleavage. Our results suggest that BLyS has the potential to serve as an excellent targeting ligand for the specific delivery of cytotoxic molecules to neoplastic B cells expressing the BLyS receptors, and that the rGel/BLyS fusion toxin may be an excellent candidate for the treatment of B-cell malignancies especially MCL and DLBCL.
Asunto(s)
Factor Activador de Células B/genética , Receptor del Factor Activador de Células B/metabolismo , Antígeno de Maduración de Linfocitos B/metabolismo , Linfocitos B/efectos de los fármacos , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión/farmacología , Proteína Activadora Transmembrana y Interactiva del CAML/metabolismo , Apoptosis/efectos de los fármacos , Receptor del Factor Activador de Células B/genética , Antígeno de Maduración de Linfocitos B/genética , Linfocitos B/metabolismo , Western Blotting , Humanos , Linfoma/tratamiento farmacológico , Linfoma/metabolismo , Linfoma/patología , ARN Mensajero/metabolismo , Proteínas Inactivadoras de Ribosomas Tipo 1 , Toxinas Biológicas/farmacología , Proteína Activadora Transmembrana y Interactiva del CAML/genética , Células Tumorales CultivadasRESUMEN
Interactions between stromal cells and tumor cells pay a major role in cancer growth and progression. This is reflected in the composition of anticancer drugs which includes compounds directed towards the immune system and tumor-vasculature in addition to drugs aimed at the cancer cells themselves. Drug-based treatment regimens are currently designed to include compounds targeting the tumor stroma in addition to the cancer cells. Treatment limiting adverse effects remains, however, one of the major challenges for drug-based therapy and novel tolerable treatment modalities with diverse high efficacy on both tumor cells and stroma is therefore of high interest. It was hypothesized that the vascular targeted fusion toxin VEGF121/rGel in combination with the intracellular drug delivery technology photochemical internalization (PCI) stimulate direct cancer parenchymal cell death in addition to inhibition of tumor perfusion, and that an immune mediated response is relevant for treatment outcome. The aim of the present study was therefore to elucidate the anticancer mechanisms of VEGF121/rGel-PCI. In contrast to VEGF121/rGel monotherapy, VEGF121/rGel-PCI was found to mediate its effect through VEGFR1 and VEGFR2, and a targeted treatment effect was shown on two VEGFR1 expressing cancer cell lines. A cancer parenchymal treatment effect was further indicated on H&E stains of CT26-CL25 and 4â¯T1 tumors. VEGF121/rGel-PCI was shown, by dynamic contrast enhanced MRI, to induce a sustained inhibition of tumor perfusion in both tumor models. A 50% complete remission (CR) of CT26.CL25 colon carcinoma allografts was found in immunocompetent mice while no CR was detected in CT26.CL25 bearing athymic mice. In conclusion, the present report indicate VEGF121/rGel -PCI as a treatment modality with multimodal tumor targeted efficacy that should be further developed towards clinical utilization.
Asunto(s)
Antineoplásicos Fitogénicos/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Proteínas Inactivadoras de Ribosomas Tipo 1/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Animales , Línea Celular Tumoral , Femenino , Luz , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/metabolismo , Receptor 1 de Factores de Crecimiento Endotelial Vascular/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Vascular endothelial growth factor-A (VEGF) exists as five different isoforms, which exert their growth stimulatory effects through interaction with the FLK and KDR receptors. The VEGF(121) isoform has been employed as a highly selective carrier of therapeutic agents to target tumor endothelial cells resulting in inhibition of tumor growth and metastasis. VEGF(121) and VEGF(121)/rGel fusion toxin containing hexa-histidine tags were expressed in Escherichia coli AD494 (DE3) pLysS. Media containing glycerol as a primary carbon source increased the specific expression levels of soluble VEGF(121) and VEGF(121)/rGel (mg/L/OD10) by more than two-fold over LB media when grown in a batchtype cultivation in a bioreactor. High cell densities over OD 40 were achieved using a fed-batch method and employing feeding medium containing glycerol and yeast extract. The overall production of the target proteins was improved 18-fold for VEGF(121) (59.2mg/L) and 27-fold for VEGF(121)/rGel (42.5mg/L), respectively, compared to the conventional flask cultivation method (3.3 and 1.6mg/L for VEGF(121) and VEGF(121)/rGel, respectively). The purified VEGF(121) and VEGF(121)/rGel fusion proteins were biologically active as assessed by phosphorylation of KDR receptors and cytotoxicity against KDR expressing cells.
Asunto(s)
Proteínas Recombinantes de Fusión/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Animales , Antineoplásicos/metabolismo , Reactores Biológicos , Células Cultivadas , Clonación Molecular , Escherichia coli/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ingeniería de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Inactivadoras de Ribosomas Tipo 1 , Porcinos , Transfección , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
We constructed a single-chain anti-gp240 antibody (designated MEL sFv) and fused this to the recombinant toxin gelonin (rGel). MEL sFv-rGel was produced in bacterial expression plasmid (pET-32), and the protein composition was confirmed by both DNA sequencing and Western analysis. Inhibition of cell-free protein synthesis by the fusion construct demonstrated an IC(50) of 100 pM, comparable with that for native gelonin (104 pM). The MEL sFv-rGel fusion toxin bound to antigen-positive but not antigen-negative cells as assessed by ELISA. Internalization into A-375 target cells was demonstrable by 1 h after exposure. Against A-375 cells, MEL sFv-rGel demonstrated an IC(50) of approximately 8 nM, which was 250-fold lower than that for free rGel (2000 nM). The cytotoxic effects of the construct did not involve apoptosis because terminal deoxynucleotidyl transferase-mediated nick end labeling assays of treated cells were negative. (125)I-labeled MEL sFv-rGel demonstrated biphasic clearance of the construct from plasma (t(1/2) alpha and t(1/2) beta were 0.46 and 7.2 h, respectively). At 72 h after administration, xenograft studies showed that the tissue:blood ratio was highest for tumor followed by spleen, kidney, and liver. Groups of tumor-bearing nude mice were treated with fusion toxin at either 2 or 20 mg/kg. Compared with saline-treated controls, for which mean tumor burden increased 6-fold, the groups treated with the high and low doses of fusion construct showed no increase or only a 2-fold increase, respectively. These studies suggest that this recombinant fusion construct has potent cytotoxic activity both in vitro and in vivo and is an excellent candidate for clinical development.
Asunto(s)
Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Inmunotoxinas/química , Inmunotoxinas/farmacología , Melanoma/tratamiento farmacológico , Melanoma/inmunología , Proteínas de Plantas/química , Proteínas de Plantas/farmacología , Animales , Antineoplásicos Fitogénicos/aislamiento & purificación , Antineoplásicos Fitogénicos/metabolismo , Apoptosis/efectos de los fármacos , Secuencia de Bases , Citotoxicidad Inmunológica , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Vectores Genéticos/genética , Humanos , Fragmentos de Inmunoglobulinas/química , Fragmentos de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/aislamiento & purificación , Fragmentos de Inmunoglobulinas/farmacología , Inmunotoxinas/genética , Inmunotoxinas/aislamiento & purificación , Etiquetado Corte-Fin in Situ , Radioisótopos de Yodo , Melanoma/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plásmidos/genética , Inhibidores de la Síntesis de la Proteína/química , Inhibidores de la Síntesis de la Proteína/aislamiento & purificación , Inhibidores de la Síntesis de la Proteína/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Proteínas Inactivadoras de Ribosomas Tipo 1 , Distribución Tisular , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The serine protease granzyme B (GrB; 25 kDa) is capable of inducing apoptosis through both caspase-dependent and caspase-independent mechanisms. We designed a novel vascular-targeting fusion construct designated as GrB/vascular endothelial growth factor (VEGF)121, which is composed of a non-heparin-binding isoform of VEGF and the proapoptotic pathway enzyme GrB fused via a short, flexible tether (G4S). The chimeric fusion gene was then cloned into a bacterial vector, and the protein was expressed in Escherichia coli and purified by nickel-NTA metal affinity chromatography. Western blotting confirmed incorporation of both VEGF121 and GrB proteins into the construct. GrB/VEGF121 specifically bound (ELISA) to porcine aortic endothelial (PAE)/FLK-1 cells overexpressing the FLK-1/KDR receptor but not to cells overexpressing the FLT-1 receptor. Immunofluoresence studies showed that the GrB moiety of GrB/VEGF121 was delivered efficiently and rapidly into the cytosol of PAE/FLK-1 cells but not into that of PAE/FLT-1 cells after 4 h treatment with GrB/VEGF121. Treatment of cells with GrB/VEGF121 showed that the IC50 was approximately 10 nM against PAE/FLK-1 cells; however, there were no cytotoxic effects observed on PAE/FLT-1 cells at doses up to 200 nM. GrB/VEGF121 induced apoptotic events specifically on PAE/FLK-1 as assessed by terminal deoxynucleotidyl transferase-mediated nick end labeling assay, DNA laddering, and cytochrome c release from mitochondria. In addition, the fusion construct mediated the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase in target endothelial cells within 4 h after treatment. In conclusion, delivery of the human proapoptotic pathway enzyme GrB to tumor vascular endothelial cells or to tumor cells may have significant therapeutic potential and represents a potent new class of targeted therapeutic agents with a unique mechanism of action.
Asunto(s)
Apoptosis , Endotelio Vascular/citología , Proteínas Proto-Oncogénicas c-bcl-2 , Proteínas Recombinantes de Fusión/farmacología , Serina Endopeptidasas/química , Toxinas Biológicas/farmacología , Factor A de Crecimiento Endotelial Vascular/química , Secuencia de Aminoácidos , Animales , Aorta/citología , Secuencia de Bases , Western Blotting , Caspasa 3 , Caspasa 8 , Caspasas/metabolismo , Clonación Molecular , Citocromos c/metabolismo , ADN/química , Fragmentación del ADN , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/patología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Vectores Genéticos , Granzimas , Humanos , Etiquetado Corte-Fin in Situ , Concentración 50 Inhibidora , Microscopía Fluorescente , Mitocondrias/metabolismo , Datos de Secuencia Molecular , Poli(ADP-Ribosa) Polimerasas/metabolismo , Isoformas de Proteínas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Porcinos , Factores de Tiempo , Proteína X Asociada a bcl-2RESUMEN
The serine protease granzyme B (GrB, 25 kDa) can initiate apoptosis by multiple mechanisms including directly activating caspases, inducing DNA fragmentation, activating the mitochondrial death pathway, and directly cleaving the nuclear matrix. The purpose of this study was to determine whether a recombinant antibody could deliver sufficient amounts of GrB to target cells to generate an apoptotic signal. The gene sequence encoding GrB was attached to the single-chain anti-melanoma antibody scFvMEL (anti-gp240) via a flexible (G(4)S) tether. The 53-kDa GrB/scFvMEL fusion protein was expressed in bacteria and purified by metal affinity chromatography. Western blotting confirmed presence of both scFvMEL and GrB proteins. The fusion construct displayed intact GrB enzymatic activity (specific activity = 2.6 x 10(5) units/ micro mol) similar to native GrB (specific activity = 4.8 x 10(5) units/ micro mol). The construct bound specifically to human A375-M melanoma cells and delivered GrB to the cytosol as assessed by confocal microscopy. Against log-phase melanoma cells, GrB/scFvMEL demonstrated an IC(50) of 20 nM and minimal cytotoxicity to non-target cells at doses of up to 1 micro M. Coadministration of exogenous perforin (PFN) to cells resulted in a slight increase in the cytotoxic effects of the GrB/scFvMEL construct on A375 target cells and a significant increase in cytotoxicity to SKBR3 (non-target) cells. The cytotoxic effects of this fusion construct on target cells were similar to those of the previously described MEL sFv/rGel fusion toxin (IC(50) approximately 20 nM). The construct produced impressive apoptotic effects by 8 h after treatment of target cells. Mediation of the apoptotic effects of GrB/scFvMEL included caspase-3 cleavage and release of cytochrome c into the cytosolic compartment from the mitochondrial compartment. These studies demonstrate that delivery of the human pro-apoptotic pathway enzyme GrB to tumor cells may have significant therapeutic potential for cancer treatment and represents a new class of targeted therapeutic agents with a defined mechanism of action.
Asunto(s)
Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Recombinantes de Fusión/farmacología , Serina Endopeptidasas/administración & dosificación , Secuencia de Bases , Línea Celular Tumoral , Clonación Molecular , Cartilla de ADN , Ensayos de Selección de Medicamentos Antitumorales , Ensayo de Inmunoadsorción Enzimática , Granzimas , Humanos , Técnicas In Vitro , Microscopía Confocal , Serina Endopeptidasas/genéticaRESUMEN
Vascular targeting for cancer is increasingly recognized as a therapeutic strategy although the lack of objective responses and the development of resistance are major limitations for clinically-available drugs. Endothelial targeted toxins exert increased toxicity compared to antiangiogenic drugs and may therefore overcome these limitations. The specificity and toxicity of targeted toxins may be increased by utilization of a drug delivery system which provides selective release of the targeted toxins in the target cells. Photochemical internalization (PCI) is a non-invasive modality which causes translocation into the cytosol of agents that are trapped in endosomes. This study describes the first use of PCI in combination with a recombinant fusion toxin targeting tumor vasculature. Endothelial cells bearing VEGFR2 treated with VEGF121/rGel showed dramatic enhancement of toxicity after PCI utilizing the photosensitizer TPCS2a (Amphinex®). We compared the PCI of VEGF121/rGel to that of bleomycin which is currently under clinical evaluation. The VEGFR2 specificity of VEGF121/rGel was shown to be preserved by the PCI treatment. PCI of VEGF121/rGel was further shown to induce vascular collapse and edema in the invasive areas of CT26.CL25 colon carcinoma tumors as shown by CD31 IHC. Antitumor effects, as assessed by tumor growth delay were found for PCI of VEGF121/rGel and PCI of bleomycin with cure rates of 40% and 33% respectively. PCI of VEGF121/rGel was, however, better tolerated compared to PCI of bleomycin. Thus, PCI of vascular targeted toxins provides higher specificity and increased tolerability compared to PCI of bleomycin and may represent an interesting clinical future for the PCI technology.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias del Colon/irrigación sanguínea , Neoplasias del Colon/tratamiento farmacológico , Sistemas de Liberación de Medicamentos/métodos , Proteínas Recombinantes de Fusión/uso terapéutico , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Animales , Antineoplásicos/administración & dosificación , Línea Celular , Línea Celular Tumoral , Colon/efectos de los fármacos , Colon/metabolismo , Colon/patología , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Femenino , Ratones , Ratones Endogámicos BALB C , Fármacos Fotosensibilizantes/administración & dosificación , Fármacos Fotosensibilizantes/uso terapéutico , Porfirinas/administración & dosificación , Porfirinas/uso terapéutico , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/genética , Porcinos , Factor A de Crecimiento Endotelial Vascular/administración & dosificación , Factor A de Crecimiento Endotelial Vascular/genéticaRESUMEN
The cytokine TWEAK and its receptor, Fn14, have emerged as potentially valuable targets for cancer therapy. Granzyme B (GrB)-containing Fn14-targeted constructs were generated containing either the Fn14 ligand TWEAK (GrB-TWEAK) or an anti-Fn14 humanized single-chain antibody (GrB-Fc-IT4) as the targeting moieties. Both constructs showed high affinity and selective cytotoxicity against a panel of Fn14-expressing human tumor cells including triple-negative breast cancer (TNBC) lines. Cellular expression of the GrB inhibitor PI-9 in target cells had no impact on the cytotoxic effect of either construct. Cellular expression of MDR1 showed no cross-resistance to the fusion constructs. GrB-TWEAK and GrB-Fc-IT4 activated intracellular caspase cascades and cytochrome c-related proapoptotic pathways consistent with the known intracellular functions of GrB in target cells. Treatment of mice bearing established HT-29 xenografts with GrB-TWEAK showed significant tumor growth inhibition compared with vehicle alone (P < 0.05). Both GrB-TWEAK and GrB-Fc-IT4 displayed significant tumor growth inhibition when administered to mice bearing orthotopic MDA-MB-231 (TNBC) tumor xenografts. The Cancer Genome Atlas analysis revealed that Fn14 mRNA expression was significantly higher in TNBC and in HER2-positive disease (P < 0.0001) compared with hormone receptor-positive breast cancer, and in basal-like 2 tumors (P = 0.01) compared with other TNBC molecular subtypes. IHC analysis of a 101 patient TNBC tumor microarray showed that 55 of 101 (54%) of tumors stained positive for Fn14, suggesting that this may be an excellent potential target for precision therapeutic approaches. Targeting Fn14 using fully human, GrB-containing fusion constructs may form the basis for a new class of novel, potent, and highly effective constructs for targeted therapeutic applications.
Asunto(s)
Receptores del Factor de Necrosis Tumoral/metabolismo , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/metabolismo , Animales , Línea Celular Tumoral , Femenino , Granzimas/genética , Granzimas/farmacología , Células HEK293 , Células HT29 , Humanos , Células Jurkat , Células MCF-7 , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Terapia Molecular Dirigida , Receptores del Factor de Necrosis Tumoral/biosíntesis , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Cadena Única/genética , Anticuerpos de Cadena Única/inmunología , Anticuerpos de Cadena Única/farmacología , Receptor de TWEAK , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The serine protease granzyme B (GrB) induces apoptosis through both caspase-dependent and -independent multiple-cascade mechanisms. VEGF121 binds to both VEGF receptor (VEGFR)-1 and VEGFR-2 receptors. We engineered a unique GrB/VEGF121 fusion protein and characterized its properties in vitro and in vivo. Endothelial and tumor cell lines showed varying levels of sensitivity to GrB/VEGF121 that correlated closely to total VEGFR-2 expression. GrB/VEGF121 localized efficiently into VEGFR-2-expressing cells, whereas the internalization into VEGFR-1-expressing cells was significantly reduced. Treatment of VEGFR-2(+) cells caused mitochondrial depolarization in 48% of cells by 48 hours. Exposure to GrB/VEGF121 induced apoptosis in VEGFR-2(+), but not in VEGFR-1(+), cells and rapid caspase activation was observed that could not be inhibited by treatment with a pan-caspase inhibitor. In vivo, GrB/VEGF121 localized in perivascular tumor areas adjacent to microvessels and in other areas in the tumor less well vascularized, whereas free GrB did not specifically localize to tumor tissue. Administration (intravenous) of GrB/VEGF121 to mice at doses up to 40 mg/kg showed no toxicity. Treatment of mice bearing established PC-3 tumor xenografts with GrB/VEGF121 showed significant antitumor effect versus treatment with GrB or saline. Treatment with GrB/VEGF121 at 27 mg/kg resulted in the regression of four of five tumors in this group. Tumors showed a two-fold lower Ki-67-labeling index compared with controls. Our results show that targeted delivery of GrB to tumor vascular endothelial cells or to tumor cells activates apoptotic cascades and this completely human construct may have significant therapeutic potential.
Asunto(s)
Granzimas/genética , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/genética , Receptor 1 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Administración Intravenosa , Animales , Apoptosis/efectos de los fármacos , Caspasas/genética , Caspasas/metabolismo , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Granzimas/administración & dosificación , Humanos , Ratones , Neoplasias/genética , Neoplasias/patología , Proteínas Recombinantes de Fusión/administración & dosificación , Receptor 1 de Factores de Crecimiento Endotelial Vascular/administración & dosificación , Receptor 2 de Factores de Crecimiento Endotelial Vascular/administración & dosificaciónRESUMEN
Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/neu-positive tumor cells. Compared with GrB/4D5, GrB/4D5/26 showed enhanced and long-lasting cellular uptake and improved delivery of GrB to the cytosol of target cells. Treatment with nanomolar concentrations of GrB/4D5/26 resulted in specific cytotoxicity, induction of apoptosis, and efficient downregulation of PI3K/Akt and Ras/ERK pathways. The endogenous presence of the GrB proteinase inhibitor 9 did not impact the response of cells to the fusion construct. Surprisingly, tumor cells resistant to lapatinib or Herceptin, and cells expressing MDR-1 resistant to chemotherapeutic agents showed no cross-resistance to the GrB-based fusion proteins. Administration (intravenous, tail vein) of GrB/4D5/26 to mice bearing BT474 M1 breast tumors resulted in significant tumor suppression. In addition, tumor tissue excised from GrB/4D5/26-treated mice showed excellent delivery of GrB to tumors and a dramatic induction of apoptosis compared with saline treatment. This study clearly showed that the completely human, functionalized GrB construct can effectively target Her2/neu-expressing cells and displays impressive in vitro and in vivo activity. This construct should be evaluated further for clinical use.
Asunto(s)
Neoplasias de la Mama/tratamiento farmacológico , Granzimas/administración & dosificación , Receptor ErbB-2/administración & dosificación , Anticuerpos de Cadena Única/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Supervivencia Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Terapia Molecular Dirigida , Fosfatidilinositol 3-Quinasas/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/inmunología , Proteínas Recombinantes de Fusión/administración & dosificación , Proteínas Recombinantes de Fusión/inmunología , Serina Proteasas/administración & dosificación , Serina Proteasas/genética , Serina Proteasas/inmunología , Transducción de Señal/efectos de los fármacos , Anticuerpos de Cadena Única/química , Anticuerpos de Cadena Única/inmunologíaRESUMEN
The TNF-like weak inducer of apoptosis (TWEAK; TNFSF12) receptor Fn14 (TNFRSF12A) is expressed at low levels in normal tissues but frequently highly expressed in a wide range of tumor types such as lung, melanoma, and breast, and therefore it is a potentially unique therapeutic target for these diverse tumor types. We have generated a recombinant protein containing a humanized, dimeric single-chain anti-fibroblast growth factor-inducible 14-kDa protein (Fn14) antibody fused to recombinant gelonin toxin as a potential therapeutic agent (designated hSGZ). The hSGZ immunotoxin is a highly potent and selective agent that kills Fn14-positive (Fn14(+)) tumor cells in vitro. Treatment of cells expressing the MDR protein MDR1 (ABCB1B) showed no cross-resistance to hSGZ. Induced overexpression of Fn14 levels in MCF7 cells through HER2 (ERBB2) signaling translated to an improved therapeutic index of hSGZ treatment. In combination with trastuzumab, hSGZ showed an additive or synergistic cytotoxic effect on HER2(+)/Fn14(+) breast cancer cell lines. Also, hSGZ treatment inhibited Erb3/Akt signaling in HER2-overexpressing breast cancer cells. Pharmacokinetic studies in mice revealed that hSGZ exhibited a biexponential clearance from plasma with a rapid initial clearance (t1/2α = 1.26 hours) followed by a seven-fold longer plasma half-life (t1/2ß = 7.29 hours). At 24, 48, and 72 hours after injection, uptake of the hSGZ into tumors was 5.1, 4.8, and 4.7%ID/g, with a tumor-to-muscle ratio of 5.6, 6.2, and 9.0, respectively. Therapeutic efficacy studies showed significant tumor inhibition effects using an MDA-MB-231/Luc breast cancer xenograft model. Our findings show that hSGZ is an effective anticancer agent and a potential candidate for clinical studies.