RESUMO
The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID50) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2. The reporter potency assay used a Vero E6 cell line engineered to constitutively express NanuLuc® luciferase, termed the VeroE6-NLuc or JM-1 cell line. Infection of JM-1 cells by a live virus, such as rVSV or measles virus, causes a cytopathic effect (CPE) and release of NanuLuc® from the cytoplasm into the supernatant, the amount of which reflects the intensity of the viral infection. The relative potency was calculated by comparison to a reference standard using parallel line analysis (PLA) in a log-log linear model. The reporter assay demonstrated good linearity, accuracy, and precision, and is therefore suitable for a vaccine potency assay. Further evaluation of the Vaccine 1 reporter assay demonstrated the robustness to a range of deliberate variation of the selected assay parameters and correlation with the plaque assay. In conclusion, we have demonstrated that the reporter assay using the JM-1 cell line could be used as a potency assay to support the manufacturing and release of multiple live virus vaccines.
RESUMO
BACKGROUND: Although the addition of epidermal growth factor receptor (EGFR) antibodies to various platinum-based chemotherapy regimens for non-small cell lung cancer (NSCLC) is being actively pursued in the clinic, rationale for the prioritization of specific regimens is lacking. MATERIALS AND METHODS: We evaluated the antitumor effects of necitumumab, a recombinant human IgG1 antibody targeting EGFR, in combination with cisplatin plus gemcitabine, pemetrexed, or paclitaxel in a panel of 9 subcutaneous tumor models of NSCLC established in nu/nu athymic mice. RESULTS: Necitumumab in combination with cisplatin/gemcitabine was particularly effective, although interestingly, the mechanisms underlying these benefits were model dependent. For example, increased tumor cell apoptosis contributed towards combination efficacy in the A549 model, in association with increased expression of hsa-miR-29b and reduced expression of antiapoptotic genes including DNA methyltransferase DNMT3B, commonly up-regulated in patients with NSCLC. Such inverse effects of combination therapy on DNMT3B and hsa-miR-29b expression were found in multiple models. Importantly, in the A549 model, hsa-miR-29b down-regulation of DMNT3b reduced promoter methylation of tumor suppressor genes such as Cell adhesion molecule 1 (CADM1), Ras associated (RalGDS/AF-6) domain family member 1 (RASSF1), and Fragile histidine triad gene (FHIT), increasing their expression. CONCLUSION: These results offer a preclinical rationale for combining an EGFR antibody with cisplatin/gemcitabine for patients with NSCLC, and provide potential molecular biomarkers for tailoring therapy.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pulmonares/tratamento farmacológico , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/imunologia , Sequência de Bases , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , Análise por Conglomerados , Metilação de DNA/efeitos dos fármacos , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Avaliação Pré-Clínica de Medicamentos , Receptores ErbB/imunologia , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Nus , MicroRNAs/genética , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Transdução de Sinais , Proteínas Supressoras de Tumor/genética , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
BACKGROUND: Rational strategies utilizing anticancer efficacy and biological principles are needed for the prioritization of specific combination targeted therapy approaches for clinical development, from among the many with experimental support. MATERIALS AND METHODS: Antibodies targeting epidermal growth factor receptor (EGFR) (cetuximab), insulin-like growth factor-1 receptor (IGF-IR) (IMC-A12) or vascular endothelial growth factor receptor 2 (VEGFR2) (DC101), were dosed alone or in combination, in 11 human tumor xenograft models established in mice. Efficacy readouts included the tumor burden and incidence of metastasis, as well as tumor active hypoxia inducible factor-1 (HIF-1), human VEGF and blood vessel density. RESULTS: Cetuximab and DC101 contributed potent and non-overlapping benefits to the combination approach. Moreover, DC101 prevented escape from IMC-A12 + cetuximab in a colorectal cancer model and cetuximab prevented escape from DC101 therapy in a pancreatic cancer model. CONCLUSION: Targeting VEGFR2 + EGFR was prioritized over other treatment strategies utilizing EGFR, IGF-IR and VEGFR2 antibodies. The criteria that proved to be valuable were a non-overlapping spectrum of anticancer activity and the prevention of resistance to another therapy in the combination.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais Humanizados , Antineoplásicos , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Quimioterapia Combinada , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Receptor IGF Tipo 1/imunologia , Receptor IGF Tipo 1/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Platelet-derived growth factor receptor beta (PDGFRbeta) is upregulated in most of solid tumors. It is expressed by pericytes/smooth muscle cells, fibroblast, macrophage, and certain tumor cells. Several PDGF receptor-related antagonists are being developed as potential antitumor agents and have demonstrated promising antitumor activity in both preclinical and clinical settings. Here, we produced a fully human neutralizing antibody, IMC-2C5, directed against PDGFRbeta from an antibody phage display library. IMC-2C5 binds to both human and mouse PDGFRbeta and blocks PDGF-B from binding to the receptor. IMC-2C5 also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules in tumor cells. In animal studies, IMC-2C5 significantly delayed the growth of OVCAR-8 and NCI-H460 human tumor xenografts in nude mice but failed to show antitumor activities in OVCAR-5 and Caki-1 xenografts. Our results indicate that the antitumor efficacy of IMC-2C5 is primarily due to its effects on tumor stroma, rather than on tumor cells directly. Combination of IMC-2C5 and DC101, an anti-mouse vascular endothelial growth factor receptor 2 antibody, resulted in significantly enhanced antitumor activity in BxPC-3, NCI-H460, and HCT-116 xenografts, compared with DC101 alone, and the trend of additive effects to DC101 treatment in several other tumor models. ELISA analysis of NCI-H460 tumor homogenates showed that IMC-2C5 attenuated protein level of vascular endothelial growth factor and basic fibroblast growth factor elevated by DC101 treatment. Finally, IMC-2C5 showed a trend of additive effects when combined with DC101/chemotherapy in MIA-PaCa-2 and NCI-H460 models. Taken together, these results lend great support to the use of PDGFRbeta antagonists in combination with other antiangiogenic agents in the treatment of a broad range of human cancers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias/tratamento farmacológico , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos/imunologia , Linhagem Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Feminino , Citometria de Fluxo , Células HCT116 , Humanos , Camundongos , Camundongos Nus , Células NIH 3T3 , Neoplasias/patologia , Biblioteca de Peptídeos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: To establish whether cetuximab, a chimeric IgG1 antibody targeting epidermal growth factor receptor, has the potential to restore responsiveness to oxaliplatin in preclinical cancer models, as has been shown with irinotecan in irinotecan refractory metastatic colorectal cancer patients. EXPERIMENTAL DESIGN: The effects of cetuximab and oxaliplatin, alone or in combination, were tested in vitro and in vivo using human colorectal cancer cell lines selected for oxaliplatin resistance, as well as parental control cell lines. Evaluations were made of subcutaneous xenograft tumor growth in nu/nu athymic mice, as well as activation of mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) and AKT, expression of DNA repair genes, density of apurinic/apyrimidinic DNA damage, and accumulation of platinum-DNA adducts in vitro. RESULTS: Oxaliplatin + cetuximab efficacy in murine subcutaneous xenograft models was greater than that of monotherapies and independent of the responsiveness to oxaliplatin monotherapy. In vitro, cetuximab reduced expression of excision repair cross-complementation group 1 and XPF, which are key components of the nucleotide excision repair pathway involved in the excision of platinum-DNA adducts. In addition, cetuximab reduced expression of XRCC1, a component of the base excision repair pathway responsible for the repair of apurinic/apyrimidinic sites. Effects of cetuximab on DNA repair protein levels were downstream to effects on mitogen-activated protein kinase and AKT pathway activation. In line with effects on DNA repair protein expression, cetuximab increased the accumulation of platinum and apurinic/apyrimidinic sites on DNA during oxaliplatin treatment. CONCLUSIONS: Cetuximab has the potential to salvage the benefits of oxaliplatin in oxaliplatin-resistant colorectal cancer patients by reducing DNA repair capacity.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Compostos Organoplatínicos/administração & dosagem , Animais , Anticorpos Monoclonais Humanizados , Western Blotting , Linhagem Celular Tumoral , Cetuximab , Neoplasias Colorretais/metabolismo , Adutos de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/fisiologia , Receptores ErbB/biossíntese , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , Citometria de Fluxo , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oxaliplatina , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Proteína 1 Complementadora Cruzada de Reparo de Raio-X , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Platelet-derived growth factor (PDGF) and its receptors (PDGFR) play important roles in tumorigenesis through stimulating tumor growth and promoting angiogenesis via enhancing pericyte recruitment and vessel maturation. Here we produced a neutralizing antibody, 1B3, directed against mouse PDGFRbeta. 1B3 binds to PDGFRbeta with high affinity (9x10(-11)M) and blocks PDGF-BB from binding to the receptor with an IC(50) of approximately 1.2 nM. The antibody also blocks ligand-stimulated activation of PDGFRbeta and downstream signaling molecules, including Akt and MAPK p42/44, in tumor cells. In animal studies, 1B3 significantly enhanced the antitumor and the anti-angiogenic activities of DC101, an antibody directed against mouse vascular endothelial growth factor receptor 2, in a pancreatic (BxPC-3) and a non-small cell lung (NCI-H460) tumor xenograft models. Treatment with the combination of 1B3 and DC101 in BxPC-3 xenograft-bearing mice resulted in tumor regression in 58% of mice compared to that in 18% of mice treated with DC101 alone. Taken together, these results lend great support to use PDGFRbeta antagonists in combinations with other antitumor and/or anti-angiogenic agents in the treatment of a variety of cancers.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Neoplasias/imunologia , Neovascularização Patológica/imunologia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/imunologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/imunologia , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/patologiaRESUMO
PURPOSE: Combination therapies that target the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor (VEGFR) pathways, are being actively tested for the treatment of cancer. In evaluating combination strategies, the ideal combination would be one in which the treatments interact in a way that is synergistic with regard to antitumor effects. Here, we have evaluated the interaction between anti-EGFR antibody Erbitux (cetuximab) and anti-VEGFR2 antibody, DC101, in preclinical models of pancreatic (BxPC-3) and colon (GEO) cancer. EXPERIMENTAL DESIGN: Analysis of the interaction between cetuximab and DC101 in vivo used a novel method for establishing the upper 95% confidence limits for the combination index (CI) of isobologram analyses, where CI < 1 indicates synergy. Assessment of tumor cell proliferation, apoptosis, VEGF production, and hypoxia, as well as tumor vascularization, was performed to gain insights into the mechanistic basis for synergy between agents targeting different tumor compartments. RESULTS: Monotherapy ED(50) values for tumor growth inhibition ranged from 1.8 to 2.3 mg/kg and 10.5 to 16.6 mg/kg for cetuximab and DC101, respectively. From the dose response of the combination treatment, it was determined that cetuximab and DC101 are synergistic in the BxPC-3 (CI = 0.1, P < 0.01) and GEO (CI = 0.1, P < 0.01) models. Overlapping effects on the tumor cell and vascular compartments form a basis for the interaction, with VEGF production and hypoxia-inducible factor 1alpha potentially acting as molecular links between EGFR and VEGFR2 inhibition. CONCLUSIONS: Results show antitumor synergy for combined EGFR and VEGFR2 targeted therapy, supporting the significant therapeutic potential of this combination strategy.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Receptores ErbB/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cetuximab , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Sinergismo Farmacológico , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Pancreáticas/patologia , Relação Estrutura-Atividade , Transplante Heterólogo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
A novel class of N-(4-{[4-(1H-benzoimidazol-2-yl)-arylamino]-methyl}-phenyl)-benzamides are described as inhibitors of the endo-beta-glucuronidase heparanase. Among them are N-(4-{[4-(1H-benzoimidazol-2-yl)-phenylamino]-methyl}-phenyl)-3-bromo-4-methoxy-benzamide (15h), and N-(4-{[5-(1H-benzoimidazol-2-yl)-pyridin-2-ylamino]-methyl}- phenyl)-3-bromo-4-methoxy-benzamide (23) which displayed good heparanase inhibitory activity (IC(50) 0.23-0.29 microM), with the latter showing oral exposure in mice.
Assuntos
Benzamidas/farmacologia , Benzimidazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Glucuronidase/antagonistas & inibidores , Administração Oral , Animais , Benzamidas/administração & dosagem , Benzamidas/química , Benzimidazóis/administração & dosagem , Benzimidazóis/química , Configuração de Carboidratos , Sequência de Carboidratos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/química , Técnicas In Vitro , Camundongos , Modelos Animais , Dados de Sequência Molecular , Estrutura Molecular , Peso Molecular , Relação Estrutura-AtividadeRESUMO
Platelet-derived growth factor receptor alpha (PDGFRalpha) is a type III receptor tyrosine kinase that is expressed on a variety of tumor types. A neutralizing monoclonal antibody to human PDGFRalpha, which did not cross-react with the beta form of the receptor, was generated. The fully human antibody, termed 3G3, has a Kd of 40 pmol/L and blocks both PDGF-AA and PDGF-BB ligands from binding to PDGFRalpha. In addition to blocking ligand-induced cell mitogenesis and receptor autophosphorylation, 3G3 inhibited phosphorylation of the downstream signaling molecules Akt and mitogen-activated protein kinase. This inhibition was seen in both transfected and tumor cell lines expressing PDGFRalpha. The in vivo antitumor activity of 3G3 was tested in human glioblastoma (U118) and leiomyosarcoma (SKLMS-1) xenograft tumor models in athymic nude mice. Antibody 3G3 significantly inhibited the growth of U118 (P=0.0004) and SKLMS-1 (P <0.0001) tumors relative to control. These data suggest that 3G3 may be useful for the treatment of tumors that express PDGFRalpha.
Assuntos
Anticorpos Monoclonais/química , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Becaplermina , Bioensaio , Linhagem Celular Tumoral , Relação Dose-Resposta Imunológica , Citometria de Fluxo , Humanos , Cinética , Ligantes , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Nus , Camundongos Transgênicos , Transplante de Neoplasias , Fosforilação , Fator de Crescimento Derivado de Plaquetas/química , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-sis , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/imunologia , Fatores de Tempo , TransfecçãoRESUMO
Andrographis paniculata plant extract is known to possess a variety of pharmacological activities. Andrographolide, the major constituent of the extract is implicated towards its pharmacological activity. We studied the cellular processes and targets modulated by andrographolide treatment in human cancer and immune cells. Andrographolide treatment inhibited the in vitro proliferation of different tumor cell lines, representing various types of cancers. The compound exerts direct anticancer activity on cancer cells by cell-cycle arrest at G0/G1 phase through induction of cell-cycle inhibitory protein p27 and decreased expression of cyclin-dependent kinase 4 (CDK4). Immunostimulatory activity of andrographolide is evidenced by increased proliferation of lymphocytes and production of interleukin-2. Andrographolide also enhanced the tumor necrosis factor-alpha production and CD marker expression, resulting in increased cytotoxic activity of lymphocytes against cancer cells, which may contribute for its indirect anticancer activity. The in vivo anticancer activity of the compound is further substantiated against B16F0 melanoma syngenic and HT-29 xenograft models. These results suggest that andrographolide is an interesting pharmacophore with anticancer and immunomodulatory activities and hence has the potential for being developed as a cancer therapeutic agent.
Assuntos
Andrographis , Antineoplásicos Fitogênicos/farmacologia , Diterpenos/farmacologia , Fitoterapia , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Western Blotting , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral/efeitos dos fármacos , Diterpenos/administração & dosagem , Diterpenos/uso terapêutico , Citometria de Fluxo , Células HT29/efeitos dos fármacos , Humanos , Interleucina-2/biossíntese , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Transplante HeterólogoRESUMO
Azadirone 1, a limonoidal constituent of Azadirachta indica is found to possess potent cytotoxic activity against a panel of human cancer cell lines in our in vitro studies. In vitro screening of a number of semi-synthetic analogues of 1 revealed that the alpha,beta-unsaturated enone moiety or its equivalent conjugated system in A-ring, C-7 acetyloxy/chloroacetyloxy or keto group in B-ring and the furan moiety are responsible for the activity of 1 and its analogues. Compound 1 and two of the semi-synthetic analogues 10 and 13 were found to possess good in vivo antitumor activity in modified hollow fiber animal models.
Assuntos
Antineoplásicos/química , Antineoplásicos/toxicidade , Azadirachta/química , Limoninas/toxicidade , Antineoplásicos/síntese química , Antineoplásicos/isolamento & purificação , Divisão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Limoninas/síntese química , Limoninas/isolamento & purificação , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
A series of 6,7-diphenyl-2,3,8,8a-tetrahydro-1H-indolizin-5-one analogues were synthesized and evaluated for cytotoxic activity against eight human cancer cell lines. Compounds 18, 21, 28, 29, 30 and 31 showed cytotoxic activity with GI(50) values in the range of 2.1-8.1 microM concentration. Among these, compounds 21 and 28 exhibited good pharmacokinetic properties. These compounds were further evaluated for their in vivo efficacy in modified hollow fibre assay (HFA).
Assuntos
Antineoplásicos/síntese química , Indolizinas/síntese química , Indolizinas/farmacologia , Animais , Antineoplásicos/farmacocinética , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Indolizinas/farmacocinética , Camundongos , Farmacocinética , Relação Estrutura-AtividadeRESUMO
In our endeavor to design and synthesize novel anticancer agents, a new series of indoloquinazoline compounds were prepared and tested initially for anticancer activity in vitro against a panel of human cancer cell lines. Most of these compounds exhibited cytotoxic activity in in vitro screens. Compounds were selected and further evaluated using a modified Hollow Fiber Assay for their preliminary in vivo activity against 12 cell lines implanted in the subcutaneous and intraperitoneal compartments in mice. The results indicate that these compounds may constitute a new class of anticancer agents.