RESUMO
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.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Genes ras , Mutação , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/metabolismo , Camptotecina/administração & dosagem , Camptotecina/análogos & derivados , Cetuximab , Neoplasias Colorretais/metabolismo , Dosagem de Genes , Genes erbB-1 , Humanos , Irinotecano , Camundongos , Camundongos Nus , Compostos Organoplatínicos/administração & dosagem , Oxaliplatina , Proteínas Proto-Oncogênicas B-raf/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Carcinoma de Células Renais/tratamento farmacológico , Indóis/farmacologia , Neoplasias Renais/tratamento farmacológico , Pirróis/farmacologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinoma de Células Renais/irrigação sanguínea , Carcinoma de Células Renais/patologia , Modelos Animais de Doenças , Interações Medicamentosas , Feminino , Humanos , Fator 1 Induzível por Hipóxia/biossíntese , Indóis/uso terapêutico , Neoplasias Renais/irrigação sanguínea , Neoplasias Renais/patologia , Vasos Linfáticos/efeitos dos fármacos , Vasos Linfáticos/patologia , Camundongos , Camundongos Nus , Mutação , Transplante de Neoplasias , Neovascularização Patológica , Pericitos/efeitos dos fármacos , Pericitos/patologia , Pirróis/uso terapêutico , Células Estromais/efeitos dos fármacos , Células Estromais/patologia , Sunitinibe , Carga Tumoral , Células Tumorais Cultivadas , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Proteína Supressora de Tumor Von Hippel-Lindau/genéticaRESUMO
Bone marrow endothelial cells (ECs) are essential for reconstitution of hematopoiesis, but their role in self-renewal of long-term hematopoietic stem cells (LT-HSCs) is unknown. We have developed angiogenic models to demonstrate that EC-derived angiocrine growth factors support in vitro self-renewal and in vivo repopulation of authentic LT-HSCs. In serum/cytokine-free cocultures, ECs, through direct cellular contact, stimulated incremental expansion of repopulating CD34(-)Flt3(-)cKit(+)Lineage(-)Sca1(+) LT-HSCs, which retained their self-renewal ability, as determined by single-cell and serial transplantation assays. Angiocrine expression of Notch ligands by ECs promoted proliferation and prevented exhaustion of LT-HSCs derived from wild-type, but not Notch1/Notch2-deficient, mice. In transgenic notch-reporter (TNR.Gfp) mice, regenerating TNR.Gfp(+) LT-HSCs were detected in cellular contact with sinusoidal ECs. Interference with angiocrine, but not perfusion, function of SECs impaired repopulation of TNR.Gfp(+) LT-HSCs. ECs establish an instructive vascular niche for clinical-scale expansion of LT-HSCs and a cellular platform to identify stem cell-active trophogens.
Assuntos
Células Endoteliais/citologia , Células Endoteliais/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Transdução de Sinais , Animais , Comunicação Celular , Linhagem da Célula , Proliferação de Células , Células Cultivadas , Técnicas de Cocultura , Meios de Cultivo Condicionados , Ligantes , Camundongos , Camundongos Knockout , Receptor Notch1/deficiência , Receptor Notch1/metabolismo , Receptor Notch2/deficiência , Receptor Notch2/metabolismoRESUMO
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.
Assuntos
Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/farmacologia , Neoplasias/tratamento farmacológico , Proteínas Serina-Treonina Quinases/imunologia , Receptores de Fatores de Crescimento Transformadores beta/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Células Matadoras Naturais/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/imunologia , Receptor do Fator de Crescimento Transformador beta Tipo II , Proteína Smad2/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cancer patients receiving epidermal growth factor receptor (EGFR) antibody therapy often experience an acneiform rash of uncertain etiology in skin regions rich in pilosebaceous units. Currently, this condition is treated symptomatically with very limited, often anecdotal success. Here, we show that a monoclonal antibody targeting murine EGFR, ME1, caused a neutrophil-rich hair follicle inflammation in mice, similar to that reported in patients. This effect was preceded by the appearance of lipid-filled hair follicle distensions adjacent to enlarged sebaceous glands. The cytokine tumor necrosis factor-alpha (TNFalpha), localized immunohistochemically to this affected region of the pilosebaceous unit, was specifically up-regulated by ME1 in skin but not in other tissues examined. Moreover, skin inflammation was reduced by cotreatment with the TNFalpha signaling inhibitor, etanercept, indicating the involvement of TNFalpha in this inflammatory process. Interleukin-1, a cytokine that frequently acts in concert with TNFalpha, is also involved in this process given the efficacy of the interleukin-1 antagonist Kineret. Our results provide a mechanistic framework to develop evidence-based trials for EGFR antibody-induced skin rash in patients with cancer.
Assuntos
Anticorpos Monoclonais/efeitos adversos , Dermatite/prevenção & controle , Receptores ErbB/imunologia , Interleucina-1/antagonistas & inibidores , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Anticorpos Monoclonais/uso terapêutico , Dermatite/etiologia , Dermatite/patologia , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/antagonistas & inibidores , Etanercepte , Exantema/induzido quimicamente , Exantema/prevenção & controle , Feminino , Humanos , Imunoglobulina G/farmacologia , Proteína Antagonista do Receptor de Interleucina 1/farmacologia , Interleucina-1/genética , Interleucina-1/metabolismo , Camundongos , Camundongos SCID , Receptores do Fator de Necrose Tumoral , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Pele/efeitos dos fármacos , Pele/metabolismo , Pele/patologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismoRESUMO
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: The objective of this study was to characterize treatment-induced circulating ligand changes during therapy with epidermal growth factor receptor (EGFR) inhibitors and evaluate their potential as surrogate indicators of the optimal biological dose. EXPERIMENTAL DESIGN: Conditioned medium from human tumor cell lines, ascites fluid from tumor xenografts, and plasma samples from normal mice, as well as colorectal cancer patients, were assessed for ligand elevations using ELISA, following treatment with cetuximab (Erbitux), an anti-mouse EGFR neutralizing antibody, or a small-molecule EGFR tyrosine kinase inhibitor. RESULTS: A rapid elevation in human transforming growth factor alpha (TGF-alpha) was observed in all cell lines after treatment with cetuximab, but not with small-molecule inhibitors. The elevation showed a dose-response effect and plateau that corresponded to the maximal decrease in A431 proliferation in vitro and HT29 tumor growth in vivo. The TGF-alpha increase was exacerbated by ongoing ligand production and cleavage from the plasma membrane but did not involve transcriptional up-regulation of TGF-alpha or the matrix metalloproteinase tumor necrosis factor-alpha-converting enzyme/ADAM17. Elevations in plasma TGF-alpha, amphiregulin, and epiregulin were also detected in normal mice treated with an anti-mouse EGFR monoclonal antibody, illustrating a host tissue-dependent component of this effect in vivo. Finally, circulating TGF-alpha increased in the plasma of six patients with EGFR-negative colorectal tumors during cetuximab treatment. CONCLUSIONS: Treatment-induced increases in circulating ligands, particularly TGF-alpha, should be serially assessed in clinical trials of anti-EGFR therapeutic antibodies as potential biomarkers to aid in determination of the optimal biological dose.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Antineoplásicos/administração & dosagem , Receptores ErbB/antagonistas & inibidores , Inibidores de Proteínas Quinases/administração & dosagem , Fator de Crescimento Transformador alfa/sangue , Animais , Anticorpos Monoclonais Humanizados , Biomarcadores/sangue , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab , Relação Dose-Resposta a Droga , Receptores ErbB/imunologia , Feminino , Humanos , Ligantes , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Transcrição Gênica , Fator de Crescimento Transformador alfa/genética , Fator de Crescimento Transformador alfa/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.
Assuntos
Medula Óssea/irrigação sanguínea , Endotélio Vascular/fisiologia , Hematopoese , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/fisiologia , Regeneração , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Ataxina-1 , Ataxinas , Vasos Sanguíneos/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/efeitos da radiação , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/biossíntese , Proteínas Nucleares/biossíntese , Deleção de Sequência , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Irradiação Corporal TotalRESUMO
Historically, the use of mouse models of metastatic disease to evaluate anticancer therapies has been hampered because of difficulties in detection and quantification of such lesions without sacrificing the mice, which in turn may also be dictated by institutional or ethical guidelines. Advancements in imaging technologies have begun to change this situation. A new method to non-invasively measure tumor burden, as yet untested to monitor spontaneous metastases, is the use of transplanted tumors expressing secretable human beta-chorionic gonadotropin (beta-hCG) that can be measured in urine. We describe examples of beta-hCG-transfected tumor cell lines for evaluating the effect of different therapies on metastatic disease, which in some cases involved monitoring tumor growth for >100 days. We used beta-hCG-tagged mouse B16 melanoma and erbB-2/Her-2-expressing human breast cancer MDA-MB-231 models, and drug treatments included metronomic low-dose cyclophosphamide chemotherapy with or without a vascular endothelial growth factor receptor 2-targeting antibody (DC101) or trastuzumab, the erbB-2/Her-2-targeting antibody. Both experimental and spontaneous metastasis models were studied; in the latter case, an increase in urine beta-hCG always foreshadowed the development of lung, liver, brain, and kidney metastases. Metastatic disease was unresponsive to DC101 or trastuzumab monotherapy treatment, as assessed by beta-hCG levels. Our results also suggest that beta-hCG levels may be set as an end point for metastasis studies, circumventing guidelines, which have often hampered the use of advanced disease models. Collectively, our data indicates that beta-hCG is an effective noninvasive preclinical marker for the long term monitoring of untreated or treated metastatic disease.
Assuntos
Gonadotropina Coriônica Humana Subunidade beta/urina , Metástase Neoplásica/patologia , Metástase Neoplásica/terapia , Vísceras/patologia , Inibidores da Angiogênese/administração & dosagem , Inibidores da Angiogênese/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Gonadotropina Coriônica Humana Subunidade beta/metabolismo , Ciclofosfamida/administração & dosagem , Ciclofosfamida/farmacologia , Progressão da Doença , Feminino , Humanos , Melanoma Experimental/patologia , Camundongos , Camundongos SCID , Receptor ErbB-2/metabolismo , Fatores de Tempo , Vísceras/efeitos dos fármacosRESUMO
PURPOSE: Major shortcomings of traditional mouse models based on xenografted human glioblastoma cell lines are that tumor cells do not invade and that genetic alterations, such as amplification of the epidermal growth factor receptor (EGFR) gene, are not maintained. Such models are thus of limited value for preclinical studies. We established a highly invasive model to evaluate the effect of antibodies against EGFR (cetuximab) and vascular endothelial growth factor receptor-2 (antibody DC101). EXPERIMENTAL DESIGN: After short-term culture, glioblastoma spheroids were implanted into the brains of nude mice. Animals were treated either i.c. with cetuximab or i.p. with DC101. Tumor burden was determined histologically using image analysis of 36 different landmark points on serial brain sections. RESULTS: Invasive xenografts were obtained from nine different glioblastomas. Three of seven cases treated with cetuximab responded with significant tumor growth inhibition, whereas four did not. All responsive tumors were derived from glioblastomas exhibiting EGFR amplification and expression of the truncated EGFRvIII variant, which were maintained in the xenografts. All nonresponsive tumors lacked EGFR amplification and EGFRvIII expression. The proportion of apoptotic cells was increased, whereas proliferation and invasion were decreased in responsive tumors. None of four xenograft cases treated with DC101 responded to treatment, and the diffusely invading tumors grew independent of angiogenesis. CONCLUSIONS: Inhibition of glioblastoma growth and invasion can be achieved using i.c. delivery of an anti-EGFR antibody, but tumor response depends on the presence of amplified and/or mutated EGFR. Antiangiogenic treatment with DC101 is not effective against diffusely invading tumors.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/prevenção & controle , Modelos Animais de Doenças , Receptores ErbB/antagonistas & inibidores , Glioblastoma/prevenção & controle , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/agonistas , Adulto , Idoso , Animais , Anticorpos Monoclonais Humanizados , Neoplasias Encefálicas/genética , Proliferação de Células , Cetuximab , Receptores ErbB/genética , Feminino , Amplificação de Genes , Glioblastoma/genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Invasividade Neoplásica , Neovascularização Patológica/prevenção & controle , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Several hypotheses have been proposed to explain how antiangiogenic drugs enhance the treatment efficacy of cytotoxic chemotherapy, including impairing the ability of chemotherapy-responsive tumors to regrow after therapy. With respect to the latter, we show that certain chemotherapy drugs, e.g., paclitaxel, can rapidly induce proangiogenic bone marrow-derived circulating endothelial progenitor (CEP) mobilization and subsequent tumor homing, whereas others, e.g., gemcitabine, do not. Acute CEP mobilization was mediated, at least in part, by systemic induction of SDF-1alpha and could be prevented by various procedures such as treatment with anti-VEGFR2 blocking antibodies or paclitaxel treatment in CEP-deficient Id mutant mice, both of which resulted in enhanced antitumor effects mediated by paclitaxel, but not by gemcitabine.
Assuntos
Inibidores da Angiogênese/uso terapêutico , Antineoplásicos/uso terapêutico , Células Endoteliais/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Células-Tronco/efeitos dos fármacos , Inibidores da Angiogênese/farmacologia , Animais , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/patologia , Neoplasias da Mama/sangue , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Carcinoma Pulmonar de Lewis/sangue , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Proliferação de Células/efeitos dos fármacos , Quimiocina CXCL12/sangue , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Quimioterapia Combinada , Células Endoteliais/patologia , Feminino , Humanos , Melanoma Experimental/sangue , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Neoplasias/sangue , Neoplasias/patologia , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Células-Tronco/patologia , Carga Tumoral/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/sangue , GencitabinaRESUMO
Vascular endothelial growth factor receptor 3 (VEGFR-3) binds VEGF-C and VEGF-D and is essential for the development of the lymphatic vasculature. Experimental tumors that overexpress VEGFR-3 ligands induce lymphatic vessel sprouting and enlargement and show enhanced metastasis to regional lymph nodes and beyond, whereas a soluble form of VEGFR-3 that blocks receptor signaling inhibits these changes and metastasis. Because VEGFR-3 is also essential for the early blood vessel development in embryos and is up-regulated in tumor angiogenesis, we wanted to determine if an antibody targeting the receptor that interferes with VEGFR-3 ligand binding can inhibit primary tumor growth. Our results show that antibody interference with VEGFR-3 function can inhibit the growth of several human tumor xenografts in immunocompromised mice. Immunohistochemical analysis showed that the blood vessel density of anti-VEGFR-3-treated tumors was significantly decreased and hypoxic and necrotic tumor tissue was increased when compared with tumors treated with control antibody, indicating that blocking of the VEGFR-3 pathway inhibits angiogenesis in these tumors. As expected, the anti-VEGFR-3-treated tumors also lacked lymphatic vessels. These results suggest that the VEGFR-3 pathway contributes to tumor angiogenesis and that effective inhibition of tumor progression may require the inhibition of multiple angiogenic targets.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias/irrigação sanguínea , Neoplasias/terapia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/antagonistas & inibidores , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Processos de Crescimento Celular , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Neovascularização Patológica/terapia , Fator C de Crescimento do Endotélio Vascular/metabolismo , Fator D de Crescimento do Endotélio Vascular/metabolismo , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The class III receptor tyrosine kinase FLT3 is expressed on the blasts of >90% of patients with B-lineage acute lymphoblastic leukemias (ALL). In addition, it is expressed at extremely high levels in ALL patients with mixed lineage leukemia rearrangements or hyperdiploidy and is sometimes mutated in these same patients. In this report, we investigate the effects of treating ALL cell lines and primary samples with human anti-FLT3 monoclonal antibodies (mAb) capable of preventing binding of FLT3 ligand. In vitro studies, examining the ability of two anti-FLT3 mAbs (IMC-EB10 and IMC-NC7) to affect FLT3 activation and downstream signaling in ALL cell lines and primary blasts, yielded variable results. FLT3 phosphorylation was consistently inhibited by IMC-NC7 treatment, but in some cell lines, IMC-EB10 actually stimulated FLT3 activation, possibly as a result of antibody-mediated receptor dimerization. Through antibody-dependent, cell-mediated cytotoxicity, such an antibody could still prove efficacious against leukemia cells in vivo. In fact, IMC-EB10 treatment significantly prolonged survival and/or reduced engraftment of several ALL cell lines and primary ALL samples in nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice. This occurred even when IMC-EB10 treatment resulted in FLT3 activation in vitro. Moreover, fluorescence-activated cell sorting and PCR analysis of IMC-EB10-treated NOD/SCID mice surviving 150 days post-leukemic cell injection revealed that FLT3 immunotherapy reduced leukemic engraftment below the level of detection in these assays (<0.001%). Furthermore, in vivo IMC-EB10 treatment did not select for resistant cells, because cells surviving IMC-EB10 treatment remain sensitive to IMC-EB10 cytotoxicity upon retransplantation. In vivo studies involving either partial depletion or activation of natural killer (NK) cells show that most of the cytotoxic effect of IMC-EB10 is mediated through NK cells. Therefore, such an antibody, either naked or conjugated to radioactive isotopes or cytotoxic agents, may prove useful in the therapy of infant ALL as well as childhood and adult ALL patients whose blasts typically express FLT3.
Assuntos
Anticorpos Monoclonais/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/terapia , Tirosina Quinase 3 Semelhante a fms/imunologia , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular Tumoral , Criança , Citotoxicidade Imunológica , Feminino , Humanos , Imunização Passiva/métodos , Células Matadoras Naturais/imunologia , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Leucemia-Linfoma Linfoblástico de Células Precursoras/imunologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Transdução de Sinais/efeitos dos fármacos , Tirosina Quinase 3 Semelhante a fms/antagonistas & inibidoresRESUMO
Vascular endothelial growth factor receptor 1 (VEGFR-1) is present on endothelial cells and subsets of human tumor cells, raising the hypothesis that angiogenic factors may promote tumor growth both by inducing angiogenesis and directly signaling through activation of VEGFR-1 on tumor cells. Here, we report that VEGFR-1 is expressed on a panel of 16 human breast tumor cell lines, and the vasculature and the tumor cell compartment of a subset of breast carcinoma lesions, and that selective signaling through VEGFR-1 on breast cancer cells supports tumor growth through downstream activation of the p44/42 mitogen-activated protein kinase (MAPK) or Akt pathways. Ligand-stimulated proliferation of breast tumor cells was inhibited by specific blockade with an anti-VEGFR-1 neutralizing monoclonal antibody. Treatment with anti-VEGFR-1 mAb significantly suppressed the growth of DU4475, MCF-7, BT-474 and MDA-MB-231 breast xenografts in athymic mice. Histological examination of anti-VEGFR-1 mAb treated tumor xenografts showed a significant reduction of activation of the p44/42 MAPK or Akt pathways in tumor cells resulting in an increase in tumor cell apoptosis. Importantly, cotreatment with mAbs targeting human VEGFR-1 on tumor cells and murine VEGFR-1 on vasculature led to more potent growth inhibition of breast tumor xenografts. The results suggest that VEGF receptors may not only modulate angiogenesis, but also directly influence the growth of VEGF receptor expressing tumors.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Anticorpos/imunologia , Neoplasias da Mama/genética , Neoplasias da Mama/imunologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Proteínas da Gravidez/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Sensibilidade e Especificidade , Transdução de Sinais , 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/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
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
Vascular endothelial growth factors (VEGF) and their receptors (VEGFR) have been implicated to play important roles in tumor-associated angiogenesis and lymphangiogenesis, and hence in tumor growth and metastasis. We previously produced a number of fully human antibodies directed against VEGF receptor 2 (VEGFR2) and VEGF receptor 3 (VEGFR3) and showed that these antibodies are capable of inhibiting growth factor (VEGF and VEGF-C)-induced receptor activation, migration, and proliferation of human endothelial cells. In this report, we constructed and produced a bispecific antibody, a diabody, using the variable domain genes of two neutralizing antibodies, IMC-1121 to VEGFR2 and hF4-3C5 to VEGFR3. The diabody binds to both VEGFR2 and VEGFR3 in a dose-dependent manner, and blocks interaction between VEGF/VEGFR2, VEGF-C/VEGFR2, and VEGF-C/VEGFR3. In cell-based assays, the diabody neutralized both VEGF and VEGF-C-stimulated activation of VEGFR2, VEGFR3, and p44/p42 mitogen-activated protein kinase in endothelial cells. Furthermore, the diabody was able to inhibit both VEGF and VEGF-C-induced migration of endothelial cells. Taken together, our results suggest that a dual blockade of both VEGFR2 and VEGFR3 simultaneously may represent a more potent approach to effective cancer therapy.
Assuntos
Anticorpos Biespecíficos/química , Imunoterapia/métodos , Neoplasias/terapia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/química , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/química , Animais , Aorta/metabolismo , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta Imunológica , Endotélio Vascular/citologia , Humanos , Fragmentos de Imunoglobulinas/química , Cinética , Sistema de Sinalização das MAP Quinases , Neoplasias/imunologia , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Veias Umbilicais/citologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 3 de Fatores de Crescimento do Endotélio Vascular/imunologiaRESUMO
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.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/farmacologia , Antineoplásicos/farmacologia , Receptores ErbB/imunologia , Receptores de Somatomedina/imunologia , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Antineoplásicos/química , Linhagem Celular Tumoral , Receptores ErbB/metabolismo , Feminino , Humanos , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/genética , Imunoglobulina G/farmacologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/patologia , Neoplasias Experimentais/terapia , Receptores de Somatomedina/metabolismo , Transdução de Sinais , Transplante HeterólogoRESUMO
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.