RESUMEN
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.
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Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/antagonistas & inhibidores , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/inmunología , Secuencia de Bases , Carcinoma de Pulmón de Células no Pequeñas/genética , Línea Celular Tumoral , Cisplatino/administración & dosificación , Análisis por Conglomerados , Metilación de ADN/efectos de los fármacos , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Evaluación Preclínica de Medicamentos , Receptores ErbB/inmunología , Femenino , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Ratones , Ratones Desnudos , MicroARNs/genética , Regiones Promotoras Genéticas , ARN Mensajero/genética , Transducción de Señal , Proteínas Supresoras de Tumor/genética , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
BACKGROUND: Clinically relevant targets for developmental drug efficacy in animal models of cancer are critical yet understudied parameters. MATERIALS AND METHODS: Cetuximab, a chimeric antibody to epidermal growth factor receptor (EGFR), was administered to athymic mice bearing subcutaneous tumors established with 13 human colorectal cancer cell lines of varying biomarker status, defined by DNA sequencing and RT-PCR. RESULTS: If tumor growth inhibition is taken as a target, as is commonly done, then in contrast to the clinical situation where KRAS mutation strongly predicts for a lack of clinically meaningful benefit in colorectal cancer patients, cetuximab alone and in combination with irinotecan-based chemotherapy were efficacious in a similar proportion of KRAS wild-type and mutant models. It was only when tumor regression was utilized to define relevant efficacy that cetuximab monotherapy was efficacious in KRAS wild-type, but not mutant models. Adding cytotoxic therapy to cetuximab treatment increased tumor regression frequency in both genotypes to the point that once again the response was similar for KRAS wild-type and mutant models. CONCLUSION: Our data support shifting the threshold for claiming clinically relevant targeted therapy efficacy in subcutaneous xenograft models towards tumor regression, rather than tumor growth inhibition, focusing on the evaluation of tumor cells that are addicted to the pathways being targeted.
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Anticuerpos Monoclonales/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Genes ras , Mutación , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Biomarcadores de Tumor/metabolismo , Camptotecina/administración & dosificación , Camptotecina/análogos & derivados , Cetuximab , Neoplasias Colorrectales/metabolismo , Dosificación de Gen , Genes erbB-1 , Humanos , Irinotecán , Ratones , Ratones Desnudos , Compuestos Organoplatinos/administración & dosificación , Oxaliplatino , Proteínas Proto-Oncogénicas B-raf/genética , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
PURPOSE: Transforming growth factor beta (TGFbeta) is a pleiotropic cytokine that affects tumor growth, metastasis, stroma, and immune response. We investigated the therapeutic efficacy of anti-TGFbeta receptor II (TGFbeta RII) antibody in controlling metastasis and tumor growth as well as enhancing antitumor immunity in preclinical tumor models. EXPERIMENTAL DESIGN: We generated neutralizing antibodies to TGFbeta RII and assessed the antibody effects on cancer, stroma, and immune cells in vitro. The efficacy and mechanism of action of the antibody as monotherapy and in combination with chemotherapy in suppression of primary tumor growth and metastasis were evaluated in several tumor models. RESULTS: Anti-TGFbeta RII antibody blocked TGFbeta RII binding to TGFbeta 1, 2, and 3, and attenuated the TGFbeta-mediated activation of downstream Smad2 kinase, invasion of cancer cells, motility of endothelial and fibroblast cells, and induction of immunosuppressive cells. Treatment with the antibody significantly suppressed primary tumor growth and metastasis and enhanced natural killer and CTL activity in tumor-bearing mice. Immunohistochemistry analysis showed cancer cell apoptosis and massive necrosis, and increased tumor-infiltrating T effector cells and decreased tumor-infiltrating Gr-1+ myeloid cells in the antibody-treated tumors. Fluorescence-activated cell sorting analysis indicated the significant reduction of peripheral Gr-1+/CD11b+ myeloid cells in treated animals. Concomitant treatment with the cytotoxic agent cyclophosphamide resulted in a significantly increased antitumor efficacy against primary tumor growth and metastasis. CONCLUSIONS: These preclinical data provide a foundation to support using anti-TGFbeta RII antibody as a therapeutic agent for TGFbeta RII-dependent cancer with metastatic capacity.
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Anticuerpos Neutralizantes/química , Anticuerpos Neutralizantes/farmacología , Neoplasias/tratamiento farmacológico , Proteínas Serina-Treonina Quinasas/inmunología , Receptores de Factores de Crecimiento Transformadores beta/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Antineoplásicos/uso terapéutico , Línea Celular Tumoral , Células Asesinas Naturales/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias/inmunología , Receptor Tipo II de Factor de Crecimiento Transformador beta , Proteína Smad2/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/inmunología , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/genética , Anticuerpos Monoclonales/inmunología , Afinidad de Anticuerpos/inmunología , Línea Celular Tumoral , Sinergismo Farmacológico , Quimioterapia Combinada , Femenino , Citometría de Flujo , Células HCT116 , Humanos , Ratones , Ratones Desnudos , Células 3T3 NIH , Neoplasias/patología , Biblioteca de Péptidos , Carga Tumoral/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Human carcinomas frequently express one or more members of the epidermal growth factor receptor family. Two family members, epidermal growth factor receptor (EGFR) and c-erbB2/neu (HER2), homodimerize or heterodimerize upon activation with ligand and trigger potent mechanisms of cellular proliferation, differentiation and migration. In this study, we examined the effect of the anti-EGFR monoclonal antibody Erbitux (cetuximab) on human tumor cells expressing both EGFR and HER2. Investigation of the effect of cetuximab on the activation of EGFR-EGFR, EGFR-HER2 and HER2-HER2 homodimers and heterodimers was conducted using the NCI-N87 human gastric carcinoma cell line. Treatment of NCI-N87 cells with cetuximab completely inhibited formation of EGFR-EGFR homodimers and EGFR-HER2 heterodimers. Activation of HER2-HER2 homodimers was not appreciably stimulated by exogenous ligand and was not inhibited by cetuximab treatment. Furthermore, cetuximab inhibited EGF-induced EGFR and HER2 phosphorylation in CAL27, NCI-H226 and NCI-N87 cells. The activation of downstream signaling molecules such as AKT, MAPK and STAT-3 were also inhibited by cetuximab in these cells. To examine the effect of cetuximab on the growth of tumors in vivo, athymic mice bearing established NCI-N87 or CAL27 xenografts were treated with cetuximab (1 mg, i.p., q3d). The growth of NCI-N87 and CAL27 tumors was significantly inhibited with cetuximab therapy compared to the control groups (p<0.0001 in both cases). In the CAL27 xenograft model, tumor growth inhibition by cetuximab treatment was similar to that by cetuximab and trastuzumab combination treatment. Immunohistological analysis of cetuximab-treated tumors showed a decrease in EGFR-HER2 signaling and reduced tumor cell proliferation. These results suggest that cetuximab may be useful in the treatment of carcinomas co-expressing EGFR and HER2.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Antineoplásicos/uso terapéutico , Dimerización , Receptores ErbB/inmunología , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Cetuximab , Citometría de Flujo , Humanos , Ratones , Ratones Desnudos , Fosforilación/efectos de los fármacos , Receptor ErbB-2/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Neoplasias Gástricas/inmunología , Neoplasias Gástricas/metabolismo , Trasplante Heterólogo , Ensayo de Tumor de Célula MadreRESUMEN
Targeted immunotherapy against tumors or angiogenesis has shown promise as an alternative approach for the treatment of malignant disease. Whether or not combining these two treatment modalities would enhance the antitumor effect was tested in mouse models of malignant melanoma. C57BL/6 mice bearing established subcutaneous B16 tumors were treated with anti-vascular endothelial growth factor receptor (anti-VEGFR) fetal liver kinase-1 (Flk-1) monoclonal antibody (mAb) DC101 and/or anti-TYRP-1/gp75 (tyrosinase-related protein-1) mAb TA99. The growth of subcutaneous B16 tumors was significantly suppressed by the mAb DC101 (63%, p<0.001) and by mAb TA99 (75%, p<0.001) treatment alone. The combined antibody (TA99+DC101) treatment resulted in a significant enhancement (93%, p<0.001) of tumor growth suppression. In a B16 pulmonary metastasis model, combined therapy with mAb DC101 and mAb TA99 resulted in a significant reduction of lung metastases compared to the control (p<0.001) and the single agent treatment groups (p<0.05). A combined modality approach that provides passive immunity to melanoma differentiation antigens as well as inhibiting tumor neovascularization may be valuable for the treatment of malignant melanoma.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Melanoma Experimental/terapia , Glicoproteínas de Membrana/inmunología , Oxidorreductasas/inmunología , Receptores de Factores de Crecimiento Endotelial Vascular/inmunología , Animales , Procesos de Crecimiento Celular/efectos de los fármacos , Melanoma Experimental/inmunología , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Metástasis de la NeoplasiaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Colorrectales/tratamiento farmacológico , Resistencia a Antineoplásicos/efectos de los fármacos , Compuestos Organoplatinos/administración & dosificación , Animales , Anticuerpos Monoclonales Humanizados , Western Blotting , Línea Celular Tumoral , Cetuximab , Neoplasias Colorrectales/metabolismo , Aductos de ADN/efectos de los fármacos , Reparación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/efectos de los fármacos , Resistencia a Antineoplásicos/fisiología , Receptores ErbB/biosíntesis , Quinasas MAP Reguladas por Señal Extracelular/efectos de los fármacos , Citometría de Flujo , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/metabolismo , Oxaliplatino , Proteínas Proto-Oncogénicas c-akt/efectos de los fármacos , Proteína 1 de Reparación por Escisión del Grupo de Complementación Cruzada de las Lesiones por Rayos X , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Mutations in the kinase domain of the epidermal growth factor receptor (EGFR) were identified in approximately 15% of all patients with non-small cell lung cancer (NSCLC). These mutations have been established as an indicator of superior response to gefitinib and erlotinib, small molecule inhibitors of the EGFR kinase domain. Whether these mutations would also render patients more susceptible to treatment with cetuximab (Erbitux), an EGFR-neutralizing antibody, is yet to be determined. In this study, we attempted to evaluate the effect of cetuximab on several NSCLC lines harboring some of the more common EGFR mutations (L858R and delL747-T753insS), as well as the recently identified kinase inhibitor-resistant mutation, T790M. We could show that the kinase activity of the abovementioned EGFR mutants was hindered by cetuximab, as detected by both cell-based phosphorylation and proliferation assays. Interestingly, cetuximab also induced enhanced degradation of the EGFR mutants as compared with the wild-type receptor. Most importantly, cetuximab successfully inhibited the growth of NSCLC lines in xenograft models. These results indicate the promising potential of cetuximab as a regimen for patients with NSCLC bearing these mutations.
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Anticuerpos Monoclonales/farmacología , Antineoplásicos/farmacología , Carcinoma de Pulmón de Células no Pequeñas/genética , Receptores ErbB/genética , Neoplasias Pulmonares/genética , Mutación/efectos de los fármacos , Animales , Anticuerpos Monoclonales Humanizados , Apoptosis , Western Blotting , Línea Celular Tumoral , Cetuximab , Dimerización , Receptores ErbB/metabolismo , Femenino , Técnica del Anticuerpo Fluorescente , Humanos , Immunoblotting , Inmunoprecipitación , Ratones , Ratones Desnudos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Ubiquitina/metabolismoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Neoplasias/inmunología , Neovascularización Patológica/inmunología , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Antineoplásicos/administración & dosificación , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Neoplasias/irrigación sanguínea , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Neovascularización Patológica/tratamiento farmacológico , Neovascularización Patológica/patologíaRESUMEN
PURPOSE: Targeting the epidermal growth factor receptor (EGFR) is a validated approach to treat cancer. In non-small cell lung cancer (NSCLC), EGFR contains somatic mutations in 10% of patients, which correlates with increased response rates to small molecule inhibitors of EGFR. We analyzed the effects of the monoclonal IgG1 antibody Erbitux (cetuximab) in NSCLC xenografts with wild-type (wt) or mutated EGFR. EXPERIMENTAL DESIGN: NSCLC cell lines were grown s.c. in nude mice. Dose-dependent efficacy was established for cetuximab. To determine whether combination therapy produces tumor regressions, cetuximab was dosed at half-maximal efficacy with chemotherapy used at maximum tolerated dose. RESULTS: Cetuximab showed antitumor activity in wt (A549, NCI-H358, NCI-H292) and mutated [HCC-827 (delE746-A750), NCI-H1975 (L858R, T790M)] EGFR-expressing xenografts. In the H292 model, cetuximab and docetaxel combination therapy was more potent to inhibit tumor growth than cetuximab or docetaxel alone. Cisplatin augmented efficacy of cetuximab to produce 6 of 10 regressions, whereas 1 of 10 regressions was found with cetuximab and no regression was found with cisplatin. Using H1975 xenografts, gemcitabine increased efficacy of cetuximab resulting in 12 of 12 regressions. Docetaxel with cetuximab was more efficacious with seven of nine regressions compared with single treatments. Cetuximab inhibited autophosphorylation of EGFR in both H292 and H1975 tumor lysates. Exploring the underlying mechanism for combination effects in the H1975 xenograft model, docetaxel in combination with cetuximab added to the antiproliferative effects of cetuximab but was the main component in this drug combination to induce apoptosis. CONCLUSIONS: Cetuximab showed antitumor activity in NSCLC models expressing wt and mutated EGFR. Combination treatments increased the efficacy of cetuximab, which may be important for the management of patients with chemorefractory NSCLC.
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Anticuerpos Monoclonales/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Receptores ErbB/metabolismo , Neoplasias Pulmonares/tratamiento farmacológico , Animales , Anticuerpos Monoclonales Humanizados , Apoptosis/efectos de los fármacos , Western Blotting , Cetuximab , Cisplatino/uso terapéutico , Docetaxel , Relación Dosis-Respuesta a Droga , Receptores ErbB/genética , Humanos , Ratones , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Taxoides/uso terapéutico , Trasplante Heterólogo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
TYRP1 (tyrosinase-related protein 1) is a melanoma antigen expressed in melanosomes and on the surface of melanoma cells. Previous studies have shown that mouse antibodies to TYRP1 localized to melanomas in vivo and inhibited tumor growth and metastasis. Here, we describe the characterization of a novel fully human anti-TYRP1 MAb (20D7) generated by immunizing HuMAb mice (Medarex). 20D7 recognized recombinant and native human TYRP1 by Western blotting and ELISA, and native TYRP1 in melanoma cells as determined by flow cytometry analysis. 20D7 cross-reacted with mouse TYRP1. The binding affinity to human TYRP1 for the human MAb was in the low nM range as determined by surface plasmon resonance kinetics. 20D7 can bind to human and mouse Fc receptor and induce a strong ADCC response against human melanoma cells in vitro. The antitumor activity of 20D7 was tested in human melanoma xenografts and mouse metastatic melanoma models in athymic nude mice. Growth of s.c. human melanoma tumors and metastatic nodules of murine B16 tumor were significantly suppressed by 20D7 compared to human IgG control. These results suggest that human anti-TYRP1 MAb may be a potent therapeutic for the treatment of malignant melanoma.
Asunto(s)
Anticuerpos Monoclonales/uso terapéutico , Anticuerpos Antineoplásicos/uso terapéutico , Melanoma Experimental/terapia , Glicoproteínas de Membrana/inmunología , Oxidorreductasas/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Anticuerpos Antineoplásicos/biosíntesis , Anticuerpos Antineoplásicos/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Línea Celular Tumoral , Proteínas del Sistema Complemento/inmunología , Femenino , Humanos , RatonesRESUMEN
PURPOSE: Vascular endothelial growth factor receptor-1 (VEGFR-1) plays important roles in promotion of tumor growth by mediating cellular functions in tumor vascular endothelium and cancer cells. Blockade of VEGFR-1 activation has been shown to inhibit pathologic angiogenesis and tumor growth, implicating VEGFR-1 as a potential therapeutic target for the treatment of cancer. We have thus developed a VEGFR-1 antagonist human monoclonal antibody designated as IMC-18F1 and evaluated its antitumor activity in preclinical experimental models to show the therapeutic potential of the antibody for cancer treatment in clinic. EXPERIMENTAL DESIGN: Human IgG transgenic mice were used for generation of anti-VEGFR-1 antibodies. Anti-VEGFR-1-specific blocking antibodies were identified using solid-phase binding and blocking assays. Inhibitory antitumor cell activity of IMC-18F1 was assessed in cell-based kinase and growth assays. Pharmacokinetic/pharmacodynamic studies were done to determine the association of antibody blood level with antitumor efficacy of the antibody in vivo. Antitumor efficacy of the anti-VEGFR-1 antibodies as monotherapy and in combination with cytotoxic agents was evaluated in human breast cancer xenograft models. RESULTS: A fully human neutralizing antibody, IMC-18F1, was shown to be a high-affinity (KD=54 pmol) inhibitor of VEGFR-1 ligand binding (VEGF-A, VEGF-B, and placental growth factor). IMC-18F1 inhibited ligand-induced intracellular activation of VEGFR-1 and mitogen-activated protein kinase signaling and prevented ligand-stimulated in vitro growth of breast cancer cells. In vivo, IMC-18F1 suppressed the growth of human breast tumor xenografts in association with reduced mitogen-activated protein kinase and Akt activation, reduced tumor cell proliferation, and increased tumor cell apoptosis. Pharmacokinetic/pharmacodynamic studies established a plasma elimination half-life of 5 days for IMC-18F1 and a steady-state trough plasma therapeutic threshold of 88 microg/mL. Importantly, inhibition of mouse and human VEGFR-1 with MF1 and IMC-18F1, respectively, enhanced the antitumor efficacy of cytotoxic agents commonly used to treat breast cancer. CONCLUSIONS: Based on preclinical validation studies, IMC-18F1 anti-VEGFR-1 has potential to provide clinical benefit to cancer patients.
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Anticuerpos Bloqueadores/uso terapéutico , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Receptores de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos Bloqueadores/sangre , Afinidad de Anticuerpos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Western Blotting , Línea Celular Tumoral , Femenino , Citometría de Flujo , Semivida , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Transgénicos , Proteínas Quinasas Activadas por Mitógenos/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular/inmunología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
RON is a member of the c-MET receptor tyrosine kinase family. Like c-MET, RON is expressed by a variety of epithelial-derived tumors and cancer cell lines and it is thought to play a functional role in tumorigenesis. To date, antagonists of RON activity have not been tested in vivo to validate RON as a potential cancer target. In this report, we used an antibody phage display library to generate IMC-41A10, a human immunoglobulin G1 (IgG1) antibody that binds with high affinity (ED50 = 0.15 nmol/L) to RON and effectively blocks interaction with its ligand, macrophage-stimulating protein (MSP; IC50 = 2 nmol/L). We found IMC-41A10 to be a potent inhibitor of receptor and downstream signaling, cell migration, and tumorigenesis. It antagonized MSP-induced phosphorylation of RON, mitogen-activated protein kinase (MAPK), and AKT in several cancer cell lines. In HT-29 colon, NCI-H292 lung, and BXPC-3 pancreatic cancer xenograft tumor models, IMC-41A10 inhibited tumor growth by 50% to 60% as a single agent, and in BXPC-3 xenografts, it led to tumor regressions when combined with Erbitux. Western blot analyses of HT-29 and NCI-H292 xenograft tumors treated with IMC-41A10 revealed a decrease in MAPK phosphorylation compared with control IgG-treated tumors, suggesting that inhibition of MAPK activity may be required for the antitumor activity of IMC-41A10. To our knowledge, this is the first demonstration that a RON antagonist and specifically an inhibitory antibody of RON negatively affects tumorigenesis. Another major contribution of this report is an extensive analysis of RON expression in approximately 100 cancer cell lines and approximately 300 patient tumor samples representing 10 major cancer types. Taken together, our results highlight the potential therapeutic usefulness of RON activity inhibition in human cancers.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/inmunología , Animales , Anticuerpos Monoclonales/inmunología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Movimiento Celular/inmunología , Células HT29 , Factor de Crecimiento de Hepatocito/antagonistas & inhibidores , Factor de Crecimiento de Hepatocito/metabolismo , Humanos , Fragmentos de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina G/farmacología , Ratones , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Células 3T3 NIH , Biblioteca de Péptidos , Fosforilación , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/química , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Animales , Becaplermina , Bioensayo , Línea Celular Tumoral , Relación Dosis-Respuesta Inmunológica , Citometría de Flujo , Humanos , Cinética , Ligandos , Sistema de Señalización de MAP Quinasas , Ratones , Ratones Desnudos , Ratones Transgénicos , Trasplante de Neoplasias , Fosforilación , Factor de Crecimiento Derivado de Plaquetas/química , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-sis , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/inmunología , Factores de Tiempo , TransfecciónRESUMEN
Both the epidermal growth factor receptor (EGFR) and the insulin-like growth factor receptor (IGFR) have been implicated in the tumorigenesis of a variety of cancers. Here we propose that simultaneous targeting of both receptors with a bispecific antibody would lead to enhanced antitumor activity. To this end, we produced a recombinant human IgG-like bispecific antibody, a Di-diabody, using the variable regions from two antagonistic antibodies: IMC-11F8 to EGFR and IMC-A12 to IGFR. The Di-diabody binds to both EGFR and IGFR and effectively blocked both EGF- and IGF-stimulated receptor activation and tumor cell proliferation. The Di-diabody also inherited the biological properties from both of its parent antibodies; it triggers rapid and significant IGFR internalization and degradation and mediates effective antibody-dependent cellular cytotoxicity in a variety of tumor cells. Finally, the Di-diabody strongly inhibited the growth of two different human tumor xenografts in vivo. Our results underscore the benefits of simultaneous targeting of two tumor targets with bispecific antibodies.
Asunto(s)
Anticuerpos Biespecíficos/biosíntesis , Anticuerpos Biespecíficos/farmacología , Antineoplásicos/farmacología , Receptores ErbB/inmunología , Receptores de Somatomedina/inmunología , Animales , Anticuerpos Biespecíficos/química , Anticuerpos Biespecíficos/genética , Antineoplásicos/química , Línea Celular Tumoral , Receptores ErbB/metabolismo , Femenino , Humanos , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/química , Inmunoglobulina G/genética , Inmunoglobulina G/farmacología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Neoplasias Experimentales/metabolismo , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Receptores de Somatomedina/metabolismo , Transducción de Señal , Trasplante HeterólogoRESUMEN
Aberrant FLT3 expression and/or mutation plays a significant role in leukemogenesis. This has prompted the development of selective small molecule tyrosine kinase inhibitors against FLT3. However, like most tyrosine kinase inhibitors, those against FLT3 are not completely specific and at the doses required to completely inhibit target, significant toxicities may occur. In addition, tyrosine kinase inhibitors for other kinases have been shown to select for cells that become resistant. To overcome some of these limitations we developed two fully human phage display monoclonal antibodies against FLT3 (IMC-EB10 and IMC-NC7). These antibodies inhibited ligand-mediated activation of wild-type FLT3 and constitutively activated mutant FLT3 and in most cell types affected downstream STAT5, AKT, and mitogen-activated protein kinase activation. In addition to interfering with FLT3 signaling, IMC-EB10 and, to a significantly lesser extent, IMC-NC7 initiated antibody-dependent cell-mediated cytotoxicity on FLT3-expressing cells. When IMC-EB10 was used in vivo to treat nonobese diabetic/severe combined immunodeficient mice given injections of primary FLT3/ITD acute myelogenous leukemia samples or myeloid cell lines with FLT3 expression, it significantly decreased engraftment of leukemic cells and increased survival, respectively. In contrast, IMC-EB10 treatment did not reduce engraftment of normal human CD34+ cord blood cells nor did it show any significant inhibition of normal murine hematopoiesis. Thus, these types of antibodies have the potential to be safe and effective new therapeutic agents for acute myelogenous leukemia and possibly other FLT3-expressing malignancies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/terapia , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales/inmunología , Citotoxicidad Celular Dependiente de Anticuerpos , Antígenos CD34/biosíntesis , Antígenos CD34/inmunología , Sangre Fetal/citología , Sangre Fetal/inmunología , Humanos , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Transducción de Señal/inmunología , Tirosina Quinasa 3 Similar a fmsRESUMEN
Recombinant protein production in plants such as corn is a promising means to generate high product yields at low comparable production cost. The anti-EGFR monoclonal antibody C225, cetuximab, is a well-characterized receptor antagonist antibody recently approved for the treatment of refractory colorectal cancer. We initiated a study to test and compare the functional activity of glycosylated and aglycosylated C225 produced in stable transgenic corn seed. Both corn antibodies were shown to be functionally indistinguishable from mammalian-derived C225 in demonstrating high-affinity binding to the EGF receptor, blocking of ligand-dependent signaling, and inhibiting cell proliferation. In addition, consistent with cetuximab, both corn antibodies possessed strong anti-tumor activity in vivo. Acute dose primate pharmacokinetic studies, however, revealed a marked increase in clearance for the glycosylated corn antibody, while the aglycosylated antibody possessed in vivo kinetics similar to cetuximab. This experimentation established that corn-derived receptor blocking monoclonal antibodies possess comparable efficacy to mammalian cell culture-derived antibody, and offer a cost effective alternative to large-scale mammalian cell culture production.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/farmacología , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/inmunología , Zea mays/genética , Zea mays/inmunología , Animales , Anticuerpos Monoclonales/farmacocinética , Anticuerpos Monoclonales Humanizados , Citotoxicidad Celular Dependiente de Anticuerpos , Antineoplásicos/farmacocinética , Cetuximab , Femenino , Humanos , Técnicas In Vitro , Cinética , Macaca fascicularis , Masculino , Ratones , Neoplasias Experimentales/inmunología , Neoplasias Experimentales/terapia , Plantas Modificadas Genéticamente , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/farmacología , Trasplante HeterólogoRESUMEN
Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. Here we show that certain "liquid" tumors such as acute myeloid leukemia not only produce VEGF but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. In addition, the leukemia-derived VEGF can also stimulate the production of growth factors, including interleukin 6 (IL6) and granulocyte-macrophage colony stimulating factor (GM-CSF), by human endothelial cells, which in turn further promotes the growth of leukemia cells (the paracrine loop). A fully human anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-2C6, strongly blocks KDR/VEGF interaction and neutralizes VEGF-stimulated activation of KDR in endothelial cells. In a system where leukemia cells are co-cultured with endothelial cells, IMC-2C6 inhibits both the production of IL6 and GM-CSF by endothelial cells and the growth of leukemia cells. Finally, IMC-2C6 effectively blocks VEGF-induced migration of KDR+ human leukemia cells, and when administered in vivo, significantly prolonged survival of mice inoculated with KDR+ human leukemia cells. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and certain types of leukemia.
Asunto(s)
Anticuerpos/farmacología , Comunicación Autocrina/efectos de los fármacos , Leucemia/tratamiento farmacológico , Leucemia/patología , Comunicación Paracrina/efectos de los fármacos , Receptor 2 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Animales , Anticuerpos/inmunología , Anticuerpos/uso terapéutico , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Regulación Neoplásica de la Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/biosíntesis , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Humanos , Interleucina-6/biosíntesis , Interleucina-6/metabolismo , Leucemia/metabolismo , Masculino , Ratones , Cordón Umbilical/inmunología , Cordón Umbilical/metabolismo , Factor A de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/farmacología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/inmunología , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
We have discovered DEGA, a novel cDNA differentially expressed in human gastric adenocarcinomas. The DEGA gene product contains a signal peptide, five leucine-rich repeat motifs and a single IgG, and transmembrane domain, suggesting its residence on the plasma membrane. Transfection of 293 cells with a DEGA-GFP fusion construct confirmed its cell surface localization. Although the cytosolic portion of the DEGA gene product does not contain known protein domains, approximately one-fifth of these residues are either a serine or a threonine, suggesting that DEGA may play a role in signal transduction. BLAST searches revealed DEGA to be an exact match to AMIGO-2, a recently identified, but functionally uncharacterized protein related to AMIGO, a leucine-rich repeat containing cell adhesion molecule implicated in axon tract development. In this report, we show that DEGA/AMIGO-2 mRNA is differentially expressed in approximately 45% of tumor versus normal tissue from gastric adenocarcinoma patients. Stable expression of a DEGA/AMIGO-2 antisense construct in the gastric adenocarcinoma cell line, AGS, led to altered morphology, increased ploidy, chromosomal instability, decreased cell adhesion/migration, and a nearly complete abrogation of tumorigenicity in nude mice. These findings suggest a potential etiologic role for DEGA/AMIGO-2 in gastric adenocarcinoma.
Asunto(s)
Adenocarcinoma/genética , Benzamidas/metabolismo , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Adhesión Celular , Movimiento Celular , Inestabilidad Cromosómica , Clonación Molecular , ADN Complementario , Humanos , Leucina , Ratones , Ratones Desnudos , Datos de Secuencia Molecular , Neoplasias Experimentales/etiología , Ploidias , Homología de Secuencia de Ácido Nucleico , Neoplasias Gástricas/patologíaRESUMEN
FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is expressed at high levels in the blasts of approximately 90% of patients with acute myelogenous leukemia (AML). Internal tandem duplications (ITDs) in the juxtamembrane domain and point mutations in the kinase domain of FLT3 are found in approximately 37% of AML patients and are associated with a poor prognosis. We report here the development and characterization of a fully human anti-FLT3 neutralizing antibody (IMC-EB10) isolated from a human Fab phage display library. IMCEB10 (immunoglobulin G1 [IgG1], kappa) binds with high affinity (KD=158 pM) to soluble FLT3 in enzyme-linked immunosorbent assay (ELISA) and to FLT3 receptor expressed on the surfaces of human leukemia cell lines. IMC-EB10 blocks the binding of FLT3 ligand (FL) to soluble FLT3 in ELISA and competes with FL for binding to cell-surface FLT3 receptor. IMC-EB10 treatment inhibits FL-induced phosphorylation of FLT3 in EOL-1 and EM3 leukemia cells and FL-independent constitutive activation of ITD-mutant FLT3 in BaF3-ITD and MV4;11 cells. Activation of the downstream signaling proteins mitogen-activated protein kinase (MAPK) and AKT is also inhibited in these cell lines by antibody treatment. The antibody inhibits FL-stimulated proliferation of EOL-1 cells and ligand-independent proliferation of BaF3-ITD cells. In both EOL-1 xenograft and BaF3-ITD leukemia models, treatment with IMC-EB10 significantly prolongs the survival of leukemia-bearing mice. No overt toxicity is observed with IMC-EB10 treatment. Taken together, these data demonstrate that IMC-EB10 is a specific and potent inhibitor of wild-type and ITD-mutant FLT3 and that it deserves further study for targeted therapy of human AML.