RESUMO
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.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Granzimas/metabolismo , Receptor de TWEAK/metabolismo , Animais , Feminino , Humanos , Camundongos , Camundongos NusRESUMO
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.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Desenvolvimento de Medicamentos , Terapia de Alvo Molecular , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Animais , Antígenos de Neoplasias/imunologia , Antineoplásicos Imunológicos/isolamento & purificação , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Expressão Gênica , Vetores Genéticos/genética , Granzimas/administração & dosagem , Granzimas/genética , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Ligação Proteica/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Sistemas de Liberação de Medicamentos , Neoplasias/tratamento farmacológico , Proteínas Inativadoras de Ribossomos Tipo 1/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Animais , Linhagem Celular Tumoral , Feminino , Luz , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Angiogenesis is a critical process in numerous diseases, and intervention in neovascularization has therapeutic value in several disease settings, including ocular diseases, arthritis, and in tumor progression and metastatic spread. Various vascular targeting agents have been developed, including those that inhibit growth factor receptor tyrosine kinases, blocking antibodies that interfere with receptor signal transduction, and strategies that trap growth factor ligands. Limited anti-tumor efficacy studies have suggested that the targeted delivery of the human pro-apoptotic molecule Granzyme B to tumor cells has significant potential for cancer treatment. Here, we review biological vascular targeting agents, and describe a unique vascular targeting agent composed of Granzyme B and the VEGF receptor ligand VEGF121. The fusion protein GrB/VEGF121 demonstrates cytotoxicity at nanomolar or sub-nanomolar levels, excellent pharmacokinetic and efficacy profiles, and has significant therapeutic potential targeting tumor vasculature.
RESUMO
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.
Assuntos
Receptores do Fator de Necrose Tumoral/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Granzimas/genética , Granzimas/farmacologia , Células HEK293 , Células HT29 , Humanos , Células Jurkat , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular , Receptores do Fator de Necrose Tumoral/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Receptor de TWEAK , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/irrigação sanguínea , Neoplasias do Colo/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Proteínas Recombinantes de Fusão/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular , Linhagem Celular Tumoral , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/patologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Fármacos Fotossensibilizantes/administração & dosagem , Fármacos Fotossensibilizantes/uso terapêutico , Porfirinas/administração & dosagem , Porfirinas/uso terapêutico , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/genética , Suínos , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
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.
Assuntos
Granzimas/genética , Neoplasias/tratamento farmacológico , Proteínas Recombinantes de Fusão/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Administração Intravenosa , Animais , Apoptose/efeitos dos fármacos , Caspases/genética , Caspases/metabolismo , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Granzimas/administração & dosagem , Humanos , Camundongos , Neoplasias/genética , Neoplasias/patologia , Proteínas Recombinantes de Fusão/administração & dosagem , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/administração & dosagem , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/administração & dosagemRESUMO
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.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Granzimas/administração & dosagem , Receptor ErbB-2/administração & dosagem , Anticorpos de Cadeia Única/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Camundongos , Terapia de Alvo Molecular , Fosfatidilinositol 3-Quinases/metabolismo , Receptor ErbB-2/genética , Receptor ErbB-2/imunologia , Proteínas Recombinantes de Fusão/administração & dosagem , Proteínas Recombinantes de Fusão/imunologia , Serina Proteases/administração & dosagem , Serina Proteases/genética , Serina Proteases/imunologia , Transdução de Sinais/efeitos dos fármacos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/imunologiaRESUMO
Fibroblast growth factor-inducible protein 14 (Fn14), the cell surface receptor for tumor necrosis factor-like weak inducer of apoptosis (TWEAK), is overexpressed in various human solid tumor types and can be a negative prognostic indicator. We detected Fn14 expression in â¼60% of the melanoma cell lines we tested, including both B-Raf WT and B-Raf(V600E) lines. Tumor tissue microarray analysis indicated that Fn14 expression was low in normal skin, but elevated in 173/190 (92%) of primary melanoma specimens and in 86/150 (58%) of melanoma metastases tested. We generated both a chemical conjugate composed of the recombinant gelonin (rGel) toxin and the anti-Fn14 antibody ITEM-4 (designated ITEM4-rGel) and a humanized, dimeric single-chain antibody of ITEM-4 fused to rGel (designated hSGZ). Both ITEM4-rGel and hSGZ were highly cytotoxic to a panel of different melanoma cell lines. Mechanistic studies showed that both immunotoxins induced melanoma cell necrosis. In addition, these immunotoxins could upregulate the cellular expression of Fn14 and trigger cell-signaling events similar to the Fn14 ligand TWEAK. Finally, treatment of mice bearing human melanoma MDA-MB-435 xenografts with either ITEM4-rGel or hSGZ showed significant tumor growth inhibition compared with controls. We conclude that Fn14 is a therapeutic target in melanoma and the hSGZ construct appears to warrant further development as a therapeutic agent against Fn14-positive melanoma.
Assuntos
Fatores Imunológicos/farmacocinética , Imunotoxinas/farmacocinética , Melanoma/tratamento farmacológico , Receptores do Fator de Necrose Tumoral/imunologia , Neoplasias Cutâneas/tratamento farmacológico , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Desenho de Fármacos , Feminino , Humanos , Fatores Imunológicos/genética , Fatores Imunológicos/imunologia , Imunotoxinas/genética , Imunotoxinas/imunologia , Melanoma/imunologia , Camundongos , Camundongos Endogâmicos BALB C , NF-kappa B/metabolismo , Ligação Proteica/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Neoplasias Cutâneas/imunologia , Receptor de TWEAK , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As a part of an ongoing assessment of its mechanism of action, we evaluated the in vivo pharmacokinetics, tissue distribution, toxicity and antitumor efficacy of VEGF(121)/rGel, a novel fusion protein. Pharmacokinetic studies showed that VEGF(121)/rGel cleared from the circulation in a biphasic manner with calculated half-lives of 0.3 and 6h for the alpha and beta phases, respectively. Pharmacokinetic evaluation of (64)Cu-DOTA-VEGF(121)/rGel showed relatively high blood retention 30 min after injection (26.6 ± 1.73% ID/g), dropping to 11.8 ± 2.83% and 0.82 ± 0.11% ID/g at 60 and 240 min post injection, respectively. Tissue uptake studies showed that kidneys, liver and tumor had the highest drug concentrations 48 h after administration. The maximum tolerated dose (MTD), based on a QOD×5 i.v. administration schedule, was found to be 18 mg/kg with an LD(50) of 25mg/kg. Treatment of BALB/c mice with VEGF(121)/rGel at doses up to the MTD caused no alterations in hematologic parameters. However, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) parameters increased in a dose-related manner. The no-observable-adverse-effect-level (NOAEL) was determined to be 20% of the MTD (3.6 mg/kg). VEGF(121)/rGel treatment of mice bearing orthotopically-placed MDA-MB-231 breast tumors caused increased vascular permeability of tumor tissue by 53% compared to saline-treated controls. Immunohistochemical analysis showed significant tumor hypoxia and necrosis as a consequence of vascular damage. In summary, VEGF(121)/rGel appears to be an effective therapeutic agent causing focused damage to tumor vasculature with minimal toxic effects to normal organs. This agent appears to be an excellent candidate for further clinical development.
Assuntos
Fator A de Crescimento do Endotélio Vascular/farmacocinética , Alanina Transaminase/metabolismo , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Aspartato Aminotransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Tecidual , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Noninvasive imaging techniques have been considered important strategies in the clinic to monitor tumor early response to therapy. In the present study, we applied RGD peptides conjugated to iron oxide nanoparticles (IONP-RGD) as contrast agents in magnetic resonance imaging (MRI) to noninvasively monitor the response of a vascular disrupting agent VEGF(121)/rGel in an orthotopic glioblastoma model. RGD peptides were firstly coupled to IONPs coated with a crosslinked PEGylated amphiphilic triblock copolymer. In vitro binding assays confirmed that cellular uptake of particles was mainly dependent on the interaction between RGD and integrin α(v)ß(3) of human umbilical vein endothelial cells (HUVEC). The tumor targeting of IONP-RGD was observed in an orthotopic U87 glioblastoma model. Finally, noninvasive monitoring of the tumor response to VEGF(121)/rGel therapy at early stages of treatment was successfully accomplished using IONP-RGD as a contrast agent for MRI, a superior method over common anatomical approaches which are based on tumor size measurements. This preclinical study can accelerate anticancer drug development and promote clinical translation of nanoprobes.
Assuntos
Compostos Férricos , Glioblastoma/tratamento farmacológico , Imageamento por Ressonância Magnética/métodos , Nanopartículas , Oligopeptídeos , Animais , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Compostos Férricos/química , Glioblastoma/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Integrina alfaVbeta3/metabolismo , Integrina beta3/metabolismo , Camundongos , Camundongos Nus , Nanopartículas/química , Nanopartículas/ultraestrutura , Neovascularização Patológica/metabolismo , Oligopeptídeos/química , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Coloração e Rotulagem , Fator A de Crescimento do Endotélio Vascular/farmacologia , Fator A de Crescimento do Endotélio Vascular/uso terapêuticoRESUMO
Therapeutic agents capable of targeting tumor cells present as established tumors and micrometastases have already demonstrated their potential in clinical trials. Immunotoxins targeting hematological malignancies and solid tumors have additionally demonstrated excellent clinical activity. This review focuses on our design and characterization studies of constructs composed of recombinant gelonin toxin fused to either growth factors or single-chain antibodies targeting solid tumor cells, tumor vasculature or hematological malignancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar levels. All of these constructs display impressive selectivity and specificity for antigen-bearing target cells in vitro and in vivo and are excellent clinical trial candidates.
Assuntos
Membrana Corioalantoide/irrigação sanguínea , Fragmentos de Imunoglobulinas/imunologia , Imunotoxinas/imunologia , Peptídeos e Proteínas de Sinalização Intercelular/imunologia , Neoplasias/tratamento farmacológico , Neovascularização Patológica , Proteínas Inativadoras de Ribossomos Tipo 1/imunologia , Anticorpos de Cadeia Única/imunologia , Animais , Antineoplásicos Fitogênicos/imunologia , Antineoplásicos Fitogênicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Linfócitos B/patologia , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Membrana Corioalantoide/metabolismo , Epitopos , Humanos , Fragmentos de Imunoglobulinas/genética , Fragmentos de Imunoglobulinas/farmacologia , Imunotoxinas/genética , Imunotoxinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Camundongos , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neoplasias/imunologia , Neoplasias/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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.
Assuntos
Inibidores da Angiogênese/farmacologia , Células Endoteliais/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Aorta/citologia , Embrião de Galinha , Galinhas , Membrana Corioalantoide/irrigação sanguínea , Membrana Corioalantoide/efeitos dos fármacos , Sistemas de Liberação de Medicamentos , Células Endoteliais da Veia Umbilical Humana , Humanos , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/genética , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Suínos , Fator A de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Current chemotherapeutics are characterized by efficient tumor cell-killing and severe side effects mostly derived from off-target toxicity. Hence targeted delivery of these drugs to tumor cells is actively sought. We previously demonstrated that poly(ethylene glycol)-functionalized carbon nanovectors are able to sequester paclitaxel, a widely used hydrophobic cancer drug, by simple physisorption and thereby deliver the drug for killing of cancer cells. The cell-killing when these drug-loaded carbon nanoparticles were used was equivalent to when a commercial formulation of paclitaxel was used. Here we show that by further mixing the drug-loaded nanoparticles with Cetuximab, a monoclonal antibody that recognizes the epidermal growth factor receptor (EGFR), paclitaxel is preferentially targeted to EGFR+ tumor cells in vitro. This supports progressing to in vivo studies. Moreover, the construct is unusual in that all three components are assembled through noncovalent interactions. Such noncovalent assembly could enable high-throughput screening of drug/antibody combinations.
Assuntos
Anticorpos Monoclonais/química , Carbono/química , Portadores de Fármacos/química , Portadores de Fármacos/metabolismo , Nanoestruturas/química , Animais , Anticorpos Monoclonais Humanizados , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cetuximab , Receptores ErbB/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Camundongos , Células NIH 3T3 , Paclitaxel/química , Paclitaxel/farmacologia , Polietilenoglicóis/químicaRESUMO
OBJECTIVES: To investigate the efficiency of magnetic resonance imaging (MRI) contrast agents employing vascular endothelial growth factor (VEGF121)/rGel conjugated MnFe2O4 nanocrystals for imaging of neovasculature using a bladder tumor model. MATERIALS AND METHODS: VEGF121/rGel was conjugated to MnFe2O4 nanoparticles (MNPs). The targeting efficiency and detection capability of the VEGF121/rGel-MNPs were investigated in both KDR-deficient (253JB-V) and KDR-overexpressing (PAE/KDR) cells using MRI. The internalization of VEGF121/rGel-MNPs into cells was confirmed by electron microscopy. Their phosphorylation ability and cytotoxicity were compared with unconjugated VEGF121/rGel. The orthotopic tumor mice were established by implanting low KDR-expressing 253JB-V cells into the bladder dome. After tail-vein injection of VEGF121/rGel-MNPs, the MR signal enhancement of intratumoral vessels by VEGF121/rGel-MNPs was observed and inhibition test using VEGF121 was also conducted. Ex vivo MR imaging of tumor tissue, and a fluorescence immunostaining study was also performed. RESULTS: The water-soluble VEGF121/rGel-MNPs (44.5 ± 1.2 nm) were stably suspended in the biologic media and exhibited a high relaxivity coefficient (423 mMs). They demonstrated sufficient targeting capability against KDR-overexpressing PAE/KDR cells, as confirmed by dose-dependent MR images and VEGF121 inhibition tests. The phosphorylation activity of KDR and cytotoxicity of VEGF121/rGel-MNPs were evaluated. VEGF121/rGel-MNPs successfully targeted the tumor and provided accurate anatomic details through (i) acquisition of clear neoangiogenic vascular distributions and (ii) obvious enhancement of the MR signal in T2*-weighted images. Immunostaining and blocking studies demonstrated the specific targeting ability of VEGF121/rGel-MNPs toward intratumoral angiogenesis. CONCLUSIONS: Synthesized VEGF121/rGel-MNPs as targeted MR imaging contrast agents can be specifically delivered to tumors and bind to KDR-expressing angiogenic tumor vessels.
Assuntos
Meios de Contraste , Compostos Férricos , Compostos de Manganês , Nanoconjugados , Proteínas Inativadoras de Ribossomos Tipo 1 , Neoplasias da Bexiga Urinária/irrigação sanguínea , Neoplasias da Bexiga Urinária/diagnóstico , Fator A de Crescimento do Endotélio Vascular , Animais , Modelos Animais de Doenças , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica , Sensibilidade e EspecificidadeRESUMO
In this study, we applied multiplexed positron emission tomography (PET) probes to monitor glucose metabolism, cellular proliferation, tumor hypoxia and angiogenesis during VEGF121/rGel therapy of breast cancer. Two doses of 12 mg/kg VEGF121/rGel, administered intraperitoneally, resulted in initial delay of tumor growth, but the growth resumed 4 days after tumor treatment was stopped. The average tumor growth rate expressed as V/V(0), were 1.11 ± 0.07, 1.21 ± 0.10, 1.58 ± 0.36 and 2.64 ± 0.72 at days 1, 3, 7 and 14, respectively. Meanwhile, the VEGF121/rGel treatment group showed V/V0 ratios of 1.04 ± 0.06, 1.05 ± 0.11, 1.09 ± 0.17 and 1.86 ± 0.36 at days 1, 3, 7 and 14, respectively. VEGF121/rGel treatment led to significantly decreased uptake of ¹8F-FPPRGD2 at day 1 (24.0 ± 8.8%, p < 0.05) and day 3 (36.3 ± 9.2%, p < 0.01), relative to the baseline, which slowly recovered to the baseline at day 14. ¹8F-FMISO uptake was increased in the treated tumors at day 1 (23.9 ± 15.7%, p < 0.05) and day 3 (51.4 ± 29.4%, p < 0.01), as compared to the control group. At days 7 and 14, ¹8F-FMISO uptake restored to the baseline level. The relative reductions in FLT uptake in treated tumors were approximately 13.0 ± 4.5% at day 1 and 25.0 ± 4.4% (p < 0.01) at day 3. No significant change of ¹8F-FDG uptake was observed in VEGF121/rGel treated tumors, compared with the control group. The imaging findings were supported by ex vivo analysis of related biomarkers. Overall, longitudinal imaging studies with 4 PET tracers demonstrated the feasibility and usefulness of multiplexed probes for quantitative measurement of antitumor effects of VEGF121/rGel at the early stage of treatment. This preclinical study should be helpful in accelerating anticancer drug development and promoting the clinical translation of molecular imaging.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Radioisótopos de Flúor , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Proteínas Inativadoras de Ribossomos Tipo 1/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Proliferação de Células/efeitos dos fármacos , Feminino , Glucose/metabolismo , Humanos , Hipóxia/tratamento farmacológico , Camundongos , Camundongos Nus , Neovascularização Patológica/prevenção & controle , Células Tumorais CultivadasRESUMO
PURPOSE: A hallmark of prostate cancer (PCa) progression is the development of osteoblastic bone metastases, which respond poorly to available therapies. We previously reported that VEGF(121)/rGel targets osteoclast precursors and tumor neovasculature. Here we tested the hypothesis that targeting nontumor cells expressing these receptors can inhibit tumor progression in a clinically relevant model of osteoblastic PCa. EXPERIMENTAL DESIGN: Cells from MDA PCa 118b, a PCa xenograft obtained from a bone metastasis in a patient with castrate-resistant PCa, were injected into the femurs of mice. Osteoblastic progression was monitored following systemic administration of VEGF(121)/rGel. RESULTS: VEGF(121)/rGel was cytotoxic in vitro to osteoblast precursor cells. This cytotoxicity was specific as VEGF(121)/rGel internalization into osteoblasts was VEGF(121) receptor driven. Furthermore, VEGF(121)/rGel significantly inhibited PCa-induced bone formation in a mouse calvaria culture assay. In vivo, VEGF(121)/rGel significantly inhibited the osteoblastic progression of PCa cells in the femurs of nude mice. Microcomputed tomographic analysis revealed that VEGF(121)/rGel restored the bone volume fraction of tumor-bearing femurs to values similar to those of the contralateral (non-tumor-bearing) femurs. VEGF(121)/rGel significantly reduced the number of tumor-associated osteoclasts but did not change the numbers of peritumoral osteoblasts. Importantly, VEGF(121)/rGel-treated mice had significantly less tumor burden than control mice. Our results thus indicate that VEGF(121)/rGel inhibits osteoblastic tumor progression by targeting angiogenesis, osteoclastogenesis, and bone formation. CONCLUSIONS: Targeting VEGF receptor (VEGFR)-1- or VEGFR-2-expressing cells is effective in controlling the osteoblastic progression of PCa in bone. These findings provide the basis for an effective multitargeted approach for metastatic PCa.
Assuntos
Neovascularização Patológica/prevenção & controle , Osteoblastos/efeitos dos fármacos , Osteoclastos/efeitos dos fármacos , Neoplasias da Próstata/tratamento farmacológico , Proteínas Recombinantes de Fusão/farmacologia , Animais , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/metabolismo , Osso e Ossos/patologia , Linhagem Celular , Linhagem Celular Tumoral , Progressão da Doença , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Técnicas de Cultura de Órgãos , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteogênese/efeitos dos fármacos , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1/genética , Proteínas Inativadoras de Ribossomos Tipo 1/metabolismo , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The transcription factor nuclear factor-κB (NF-κB) is a central mediator of growth and homeostasis for both normal and neoplastic cells. IκBα is the natural intracellular inhibitor of NF-κB and can effectively complex with and thereby inhibit the biologic activity and translocation of NF-κB to the nucleus. We designed a fusion protein designated IκBα/scFvMEL composing of human IκBα and the single-chain antibody scFvMEL, targets melanoma gp240 antigen. Cells treated with IκBα/scFvMEL before irradiation showed specifically inhibition of both constitutive and radiation-induced NF-κB activity on gp240 antigen-positive A375M cells. Pretreatment of A375M cells with IκBα/scFvMEL significantly sensitized melanoma cells to ionizing radiation assessed using a clonogenic survival assay. Mechanistic studies showed that IκBα/scFvMEL, when exogenously added to A375M cells, could be coimmunoprecipitated with the p65 subunit of NF-κB. IκBα/scFvMEL inhibited in a time and/or dose-dependent manner of tumor necrosis factor α- or radiation-induced NF-κB activity in vitro. IκBα/scFvMEL was also shown to specifically inhibit the translocation of the NF-κB p65 subunit to the cell nucleus and NF-κB-mediated gene transcription. Further, initial studies showed that mice bearing well-established A375M xenografts were treated (intravenously) with IκBα/scFvMEL and showed a significant suppression of tumor growth. We also observed a decrease in levels of Bcl-2 and Bcl-XL signaling events downstream of NF-κB in the tumor model. These studies demonstrate for the first time that tumor cell-targeted delivery of IκBα may be beneficial for the treatment of melanoma when combined with standard anticancer therapies such as radiation.
Assuntos
Proteínas I-kappa B/uso terapêutico , Melanoma Experimental/terapia , NF-kappa B/antagonistas & inibidores , Proteoglicanas/imunologia , Radiossensibilizantes/farmacologia , Proteínas Recombinantes de Fusão/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , Animais , Apoptose , Western Blotting , Núcleo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Ensaio de Desvio de Mobilidade Eletroforética , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Nus , Inibidor de NF-kappaB alfa , Fosforilação , Transporte Proteico/efeitos da radiação , RNA Mensageiro/genética , Radiação Ionizante , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/efeitos da radiação , Fator de Transcrição RelA/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
UNLABELLED: Vascular endothelial growth factor A (VEGF-A) and its receptors, Flt-1/FLT-1 (VEGFR-1) and Flk-1/KDR (VEGFR-2), are key regulators of tumor angiogenesis and tumor growth. The purpose of this study was to determine the antiangiogenic and antitumor efficacies of a vasculature-targeting fusion toxin (VEGF(121)/rGel) composed of the VEGF-A isoform VEGF(121) linked with a G(4)S tether to recombinant plant toxin gelonin (rGel) in an orthotopic glioblastoma mouse model by use of noninvasive in vivo bioluminescence imaging (BLI), MRI, and PET. METHODS: Tumor-bearing mice were randomized into 2 groups and balanced according to BLI and MRI signals. PET with (64)Cu-1,4,7,10-tetraazacyclododedane-N,N',N'',N'''-tetraacetic acid (DOTA)-VEGF(121)/rGel was performed before VEGF(121)/rGel treatment. (18)F-Fluorothymidine ((18)F-FLT) scans were obtained before and after treatment to evaluate VEGF(121)/rGel therapeutic efficacy. In vivo results were confirmed with ex vivo histologic and immunohistochemical analyses. RESULTS: Logarithmic transformation of peak BLI tumor signal intensity revealed a strong correlation with MRI tumor volume (r = 0.89, n = 14). PET with (64)Cu-DOTA-VEGF(121)/rGel before treatment revealed a tumor accumulation (mean +/- SD) of 11.8 +/- 2.3 percentage injected dose per gram at 18 h after injection, and the receptor specificity of the tumor accumulation was confirmed by successful blocking of the uptake in the presence of an excess amount of VEGF(121). PET with (18)F-FLT revealed significant a decrease in tumor proliferation in VEGF(121)/rGel-treated mice compared with control mice. Histologic analysis revealed specific tumor neovasculature damage after treatment with 4 doses of VEGF(121)/rGel; this damage was accompanied by a significant decrease in peak BLI tumor signal intensity. CONCLUSION: The results of this study suggest that future clinical multimodality imaging and therapy with VEGF(121)/rGel may provide an effective means to prospectively identify patients who will benefit from VEGF(121)/rGel therapy and then stratify, personalize, and monitor treatment to obtain optimal survival outcomes.
Assuntos
Glioblastoma/tratamento farmacológico , Proteínas de Plantas/uso terapêutico , Proteínas Recombinantes de Fusão/uso terapêutico , Fator A de Crescimento do Endotélio Vascular/uso terapêutico , Animais , Barreira Hematoencefálica , Radioisótopos de Cobre , Didesoxinucleosídeos , Glioblastoma/diagnóstico , Imageamento por Ressonância Magnética , Camundongos , Proteínas de Plantas/metabolismo , Tomografia por Emissão de Pósitrons , Receptores de Fatores de Crescimento do Endotélio Vascular/análise , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Inativadoras de Ribossomos Tipo 1 , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.