RESUMO
Fibroblast growth factors (FGFs) are important regulators of hematopoiesis and have been implicated in the tumorigenesis of solid tumors. Recent evidence suggests that FGF signaling through FGF receptors (FGFRs) may play a role in the proliferation of subsets of acute myeloid leukemias (AMLs). However, the precise mechanism and specific FGF receptors that support leukemic cell growth are not known. We show that FGF-2, through activation of FGFR1beta signaling, promotes survival, proliferation and migration of AML cells. Stimulation of FGFR1beta results in phosphoinositide 3-kinase (PI3-K)/Akt activation and inhibits chemotherapy-induced apoptosis of leukemic cells. Neutralizing FGFR1-specific antibody abrogates the physiologic and chemoprotective effects of FGF-2/FGFR1beta signaling and inhibits tumor growth in mice xenotransplanted with human AML. These data suggest that activation of FGF-2/FGFR1beta supports progression and chemoresistance in subsets of AML. Therefore, FGFR1 targeting may be of therapeutic benefit in subsets of AML.
Assuntos
Movimento Celular , Resistencia a Medicamentos Antineoplásicos , Leucemia Mieloide/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Doença Aguda , Idoso de 80 Anos ou mais , Proteína Relacionada com Agouti , Animais , Anticorpos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/genética , Masculino , Camundongos , Camundongos SCID , Fosforilação , Subunidades Proteicas/efeitos dos fármacos , Subunidades Proteicas/metabolismo , RNA Mensageiro/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Transdução de Sinais/efeitos dos fármacos , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Vascular endothelial growth factor (VEGF) and its receptors (VEGFR) have been implicated in promoting solid tumor growth and metastasis via stimulating tumor-associated angiogenesis. We recently showed that certain 'liquid' tumors such as leukemia not only produce VEGF, but also express functional VEGFR, resulting in an autocrine loop for tumor growth and propagation. A chimeric anti-VEGFR2 (or kinase insert domain-containing receptor, KDR) antibody, IMC-1C11, was shown to be able to inhibit VEGF-induced proliferation of human leukemia cells in vitro, and to prolong survival of nonobese diabetic-severe combined immune deficient (NOD-SCID) mice inoculated with human leukemia cells. Here we produced two fully human anti-KDR antibodies (IgG1), IMC-2C6 and IMC-1121, from Fab fragments originally isolated from a large antibody phage display library. These antibodies bind specifically to KDR with high affinities: 50 and 200 pM for IMC-1121 and IMC-2C6, respectively, as compared to 270 pM for IMC-1C11. Like IMC-1C11, both human antibodies block VEGF/KDR interaction with an IC(50) of approximately 1 nM, but IMC-1121 is a more potent inhibitor to VEGF-stimulated proliferation of human endothelial cells. These anti-KDR antibodies strongly inhibited VEGF-induced migration of human leukemia cells in vitro, and when administered in vivo, significantly prolonged survival of NOD-SCID mice inoculated with human leukemia cells. It is noteworthy that the mice treated with antibody of the highest affinity, IMC-1121, survived the longest period of time, followed by mice treated with IMC-2C6 and IMC-1C11. Taken together, our data suggest that anti-KDR antibodies may have broad applications in the treatment of both solid tumors and leukemia. It further underscores the efforts to identify antibodies of high affinity for enhanced antiangiogenic and antitumor activities.
Assuntos
Anticorpos Monoclonais/farmacologia , Leucemia Experimental/tratamento farmacológico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/imunologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Afinidade de Anticorpos , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Feminino , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Leucemia Experimental/mortalidade , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Proteínas Recombinantes de Fusão , Taxa de Sobrevida , Veias Umbilicais/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
High throughput screening (HTS) of large compound libraries for inhibitors of growth factors raises the requirement for simple yet reliable assays. Fibroblast growth factors (FGFs) play a pivotal role in the multistep pathway of malignant transformation, tumor progression, metastasis, and angiogenesis. FGF-2 (basic FGF) requires a cooperative interaction with heparin or heparan sulfate proteoglycans in order to form functional growth factor-receptor complexes that are essential for receptor binding and activation. We have developed a simple screening system, devised to identify molecules that modulate heparin-FGF-receptor interactions. The system is composed of a heparin matrix, FGF-2, and a FGF receptor-1 protein engineered by genetically fusing the extracellular domain of FGF receptor-1 to alkaline phosphatase (FRAP). The screen is conducted using 96-well plates to which heparin has been covalently attached. FGF-2 is then bound to the plates through heparin-FGF interactions, followed by the addition of FRAP and compounds to be screened for modulation of heparin-FGF, receptor-heparin, and receptor-FGF interactions. The endpoint of the assay is measured enzymatically using the alkaline phosphatase (AP)-catalyzed formation of a chromogenic product, which is directly proportional to the amount of FRAP present on the plates as a heparin-FGF-FRAP ternary complex. Reduced AP values relative to control, as measured by spectrophotometry, indicate inhibition of the formation of an active FGF-receptor-heparin complex. The simple and versatile nature of the assay makes it an attractive HTS system. The screen has identified several potent inhibitors of FGF-2 receptor binding and activation. Furthermore, secondary screening of the HTS-recognized compounds identified several compounds that have the capacity to block growth factor-mediated tumor progression and angiogenesis in vivo.
Assuntos
Avaliação Pré-Clínica de Medicamentos/métodos , Heparina/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Aorta , Automação , Células CHO , Bovinos , Células Cultivadas , Cricetinae , Relação Dose-Resposta a Droga , Endotélio/citologia , Endotélio/metabolismo , Fator 2 de Crescimento de Fibroblastos/antagonistas & inibidores , Fator 2 de Crescimento de Fibroblastos/metabolismo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Reprodutibilidade dos TestesRESUMO
Vascular endothelial growth factor (VEGF) binds to and mediates its activity mainly through two tyrosine kinase receptors, VEGF receptor 1 [or fms-like tyrosine kinase receptor (Flt-1)] and VEGF receptor 2 [or kinase insert domain-containing receptor (KDR)]. Numerous studies have shown that overexpression of VEGF and its receptor plays an important role in tumor-associated angiogenesis and hence in both tumor growth and metastasis. We demonstrated previously that antagonistic antibodies to KDR specifically inhibited VEGF-stimulated receptor activation, cell migration, and endothelial cell mitogenesis. Here we constructed a recombinant bifunctional diabody that is capable of blocking both Flt-1 and KDR from binding to their ligands, including VEGF and placenta growth factor (PlGF). The diabody was expressed in Escherichia coli and purified by single-step affinity chromatography. The diabody retained the capacity to bind both KDR and Flt-1 and effectively blocked interaction between KDR and VEGF, Flt-1 and VEGF, and Flt-1 and PlGF. Furthermore, the diabody is a stronger inhibitor than its parent antibodies to VEGF-stimulated mitogenesis of human endothelial cells, as well as both VEGF- and PlGF-induced migration of human leukemia cells. Taken together, our results suggest that dual receptor blockade with the bifunctional diabody may prove to be a more efficient approach in inhibiting VEGF-stimulated angiogenesis.
Assuntos
Anticorpos Biespecíficos/imunologia , Fatores de Crescimento Endotelial/antagonistas & inibidores , Linfocinas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Células 3T3 , Animais , Anticorpos Biespecíficos/genética , Anticorpos Biespecíficos/metabolismo , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Clonagem Molecular , Fatores de Crescimento Endotelial/metabolismo , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Células HL-60/citologia , Humanos , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/genética , Cadeias Leves de Imunoglobulina/imunologia , Linfocinas/metabolismo , Linfocinas/farmacologia , Camundongos , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/genética , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Using an orthotopic intracerebral model, we investigated whether systemic treatment with DC101, a monoclonal antibody against vascular endothelial growth factor receptor (VEGFR)-2, could inhibit angiogenesis and the growth of human glioblastoma cells in severe combined immunodeficient mice. Intraperitoneal treatment with DC101, control IgG, or PBS was initiated either on day 0 or, in another series, on day 6 after tumor cell implantation, and animals were killed approximately 2 weeks after tumor cell injection. Tumor volumes in animals treated with DC101 were reduced by 59 and 81% compared with IgG and PBS controls, respectively (P < 0.001), when treatment was initiated immediately, and similar results were obtained when treatment started on day 6. Microvessel density in tumors of DC101-treated animals was reduced by at least 40% compared with animals treated with control IgG or PBS (P < 0.01). We observed a reduction in tumor cell proliferation and an increase in apoptosis in DC101-treated animals (P < 0.001). However, in mice treated with DC101, we also noticed a striking increase in the number and total area of small satellite tumors clustered around, but distinct from, the primary. These satellites usually contained central vessel cores, and tumor cells often had migrated over long distances along the host vasculature to eventually reach the surface and spread leptomeningeally. We conclude that systemic antagonization of VEGFR-2 can inhibit glioblastoma neovascularization and growth but can lead to increased cooption of preexistent cerebral blood vessels. Therefore, a combination of different treatment modalities which also include anti-invasive therapy may be needed for an effective therapy against glioblastoma, and the use of an antibody against VEGFR-2 may be one effective component.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias Encefálicas/irrigação sanguínea , Glioblastoma/irrigação sanguínea , Neovascularização Patológica/prevenção & controle , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/terapia , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Feminino , Glioblastoma/patologia , Glioblastoma/terapia , Humanos , Camundongos , Camundongos Nus , Camundongos SCID , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/biossíntese , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/biossíntese , Receptores de Fatores de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Antiangiogenic agents block the effects of tumor-derived angiogenic factors (paracrine factors), such as vascular endothelial growth factor (VEGF), on endothelial cells (EC), inhibiting the growth of solid tumors. However, whether inhibition of angiogenesis also may play a role in liquid tumors is not well established. We recently have shown that certain leukemias not only produce VEGF but also selectively express functional VEGF receptors (VEGFRs), such as VEGFR-2 (Flk-1, KDR) and VEGFR1 (Flt1), resulting in the generation of an autocrine loop. Here, we examined the relative contribution of paracrine (EC-dependent) and autocrine (EC-independent) VEGF/VEGFR signaling pathways, by using a human leukemia model, where autocrine and paracrine VEGF/VEGFR loops could be selectively inhibited by neutralizing mAbs specific for murine EC (paracrine pathway) or human tumor (autocrine) VEGFRs. Blocking either the paracrine or the autocrine VEGF/VEGFR-2 pathway delayed leukemic growth and engraftment in vivo, but failed to cure inoculated mice. Long-term remission with no evidence of disease was achieved only if mice were treated with mAbs against both murine and human VEGFR-2, whereas mAbs against human or murine VEGFR-1 had no effect on mice survival. Therefore, effective antiangiogenic therapies to treat VEGF-producing, VEGFR-expressing leukemias may require blocking both paracrine and autocrine VEGF/VEGFR-2 angiogenic loops to achieve remission and long-term cure.
Assuntos
Comunicação Autócrina , Fatores de Crescimento Endotelial/metabolismo , Leucemia Promielocítica Aguda/fisiopatologia , Linfocinas/metabolismo , Comunicação Parácrina , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Transdução de Sinais , Animais , Divisão Celular , Células Cultivadas , Técnicas de Cocultura , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Células HL-60 , Humanos , Leucemia Promielocítica Aguda/metabolismo , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Neoplasias Experimentais/metabolismo , Neoplasias Experimentais/fisiopatologia , Neovascularização Patológica/fisiopatologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Fatores de Tempo , Transplante Heterólogo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Inhibitors of epidermal growth factor receptor (EGFR) signaling are among the novel drugs showing great promise for cancer treatment in the clinic. However, the possibility of acquired resistance to such drugs because of tumor cell genetic instabilities has not yet been explored. Here we report the experimental derivation and properties of such cell variants obtained from recurrent tumor xenografts of the human A431 squamous cell carcinoma, after two consecutive cycles of therapy with one of three different anti-EGFR monoclonal antibodies: mR3, hR3, or C225. Initial response to a 2-week period of treatment was generally total tumor regression and was not significantly different among the three antibody groups. However, tumors often reappeared at the site of inoculation, generally after prolonged latency periods, and most of the tumors became refractory to a second round of therapy. Cell lines established from such resistant tumors retained high EGFR expression, normal sensitivity to anti-EGFR antibody or ligand, and unaltered growth rate when compared with the parental line in vitro. In contrast, the A431 cell variants exhibited an accelerated growth rate and a significantly attenuated response to anti-EGFR antibodies in vivo relative to the parental line. Because of the reported suppressive effect of EGFR inhibitors on vascular endothelial growth factor (VEGF) expression, and the demonstrated role of VEGF in the angiogenesis and growth of A431 tumor xenografts, relative VEGF expression was examined. Five of six resistant variants expressed increased levels of VEGF, which paralleled an increase in both angiogenic potential in vitro and tumor angiogenesis in vivo. In addition, elevated expression of VEGF in variants of A431 cells obtained by gene transfection rendered the cells significantly resistant to anti-EGFR antibodies in vivo. Taken together, the results suggest that, at least in the A431 system, variants displaying acquired resistance to anti-EGFR antibodies can emerge in vivo and can do so, at least in part, by mechanisms involving the selection of tumor cell subpopulations with increased angiogenic potential.
Assuntos
Anticorpos Monoclonais/farmacologia , Carcinoma de Células Escamosas/irrigação sanguínea , Receptores ErbB/antagonistas & inibidores , Neovascularização Patológica/patologia , Animais , Anticorpos Monoclonais/imunologia , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/terapia , Resistencia a Medicamentos Antineoplásicos , Fatores de Crescimento Endotelial/biossíntese , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/fisiologia , Receptores ErbB/imunologia , Humanos , Linfocinas/biossíntese , Linfocinas/genética , Linfocinas/fisiologia , Camundongos , Camundongos SCID , Recidiva Local de Neoplasia , Neovascularização Patológica/metabolismo , Células Tumorais Cultivadas , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio Vascular , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Adult mammalian angiogenesis occurs predominantly in female reproductive organs: the ovary and the uterus. Angiogenesis is very active during corpus luteum formation. A key regulator of angiogenesis is vascular endothelial growth factor (VEGF), which is highly expressed during corpus luteum formation. Inhibition of VEGF activity can block the formation and function of the corpora lutea by preventing angiogenesis. The VEGF receptor 2 (VEGF-R2) mediates the angiogenic action of VEGF and is expressed during corpus luteum formation. We hypothesized that treatment with an antibody against VEGF-R2 would inhibit luteal angiogenesis by blocking VEGF/VEGF-R2 interaction. Immature mice were induced to superovulate with PMSG/hCG resulting in neovascularization in the corpora lutea, as evidenced by abundant staining for the endothelial-specific adhesion molecule PECAM. Multiple doses of a monoclonal antibody against the VEGF-R2 (DC101) were administered to immature mice. Treatment was initiated 2 days prior to the induction of superovulation with PMSG/hCG. This antibody inhibited luteal angiogenesis as evidenced by the lack of PECAM staining in the center of the corpora lutea. Multiple dose treatment with antibody initiated prior to gonadotropin administration could not dissociate the luteal inhibition from the consequences of inhibition of angiogenesis in the developing follicle. Administration of a single, preovulatory dose of anti-VEGF-R2 antibody, such that follicular angiogenesis would not be affected, also inhibited luteal development, demonstrating that luteal angiogenesis is required for corpus luteal development. We conclude that VEGF acting through VEGF-R2 has an obligatory role in luteal angiogenesis and corpus luteum formation.
Assuntos
Corpo Lúteo/irrigação sanguínea , Neovascularização Fisiológica , Ovulação/fisiologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Envelhecimento , Animais , Anticorpos/farmacologia , Corpo Lúteo/efeitos dos fármacos , Corpo Lúteo/fisiologia , Fatores de Crescimento Endotelial/metabolismo , Feminino , Rim/metabolismo , Linfocinas/metabolismo , Camundongos , Ovário/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Progesterona/sangue , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
VDUP1 encodes a vitamin D3-inducible gene product that has been shown to be down-regulated in chemically-induced mammary tumors in rats. It has recently been reported to negatively regulate thioredoxin expression and function. We have cloned the mouse VDUP1 gene and characterized its genomic locus. The VDUP1 coding region spans eight exons within a total length of 2.3 kb located on mouse chromosome 3. Consensus sites for polyadenylation were identified 1.3 kb downstream of the gene, defining a long 3' untranslated region. The minimal functional VDUP1 promoter contains TATA and CCAAT boxes and transcription is initiated from two major start sites downstream. A direct repeat element located proximal to the TATA with homology to the USF binding site was identified as a potential regulator of VDUP1gene expression. Expression analysis determined that VDUP1 mRNA was markedly induced in myeloma cells in high density cell culture, but not in sub-confluent cells arrested by serum deprivation. All samples of a panel of mouse immortalized or transformed cell lines were shown to express abundant levels of VDUP1 mRNA.
Assuntos
Proteínas de Transporte/genética , Tiorredoxinas , Animais , Sequência de Bases , Northern Blotting/métodos , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Análise de Sequência de DNARESUMO
Antibodies represent a unique class of therapeutics because of their high specificity towards a defined target antigen. Recent clinical success with antibody-based cancer therapeutics has led to an increase in the clinical development of these agents. Antibody therapeutics offer a promising approach for inhibiting new blood vessel formation (angiogenesis), which is associated with a variety of diseases, including cancer. In this review we will focus on angiogenesis-related mechanisms targeted by antibody-based therapeutics, with an emphasis on those studies where pre-clinical in vivo data is available.
RESUMO
Vascular endothelial cadherin (VE-cadherin) is an endothelial cell-specific cadherin that plays an important role in the control of vascular organization. Blocking VE-cadherin antibodies strongly inhibit angiogenesis, and inactivation of VE-cadherin gene causes embryonic lethality due to a lack of correct organization and remodeling of the vasculature. Hence, inhibitors of VE-cadherin adhesive properties may constitute a tool to prevent tumor neovascularization. In this paper, we tested different monoclonal antibodies (mAbs) directed to human VE-cadherin ectodomain for their functional activity. Three mAbs (Cad 5, BV6, BV9) were able to increase paracellular permeability, inhibit VE-cadherin reorganization, and block angiogenesis in vitro. These mAbs could also induce endothelial cell apoptosis in vitro. Two additional mAbs, TEA 1.31 and Hec 1.2, had an intermediate or undetectable activity, respectively, in these assays. Epitope mapping studies show that BV6, BV9, TEA 1.31, and Hec 1.2 bound to a recombinant fragment spanning the extracellular juxtamembrane domains EC3 through EC4. In contrast, Cad 5 bound to the aminoterminal domain EC1. By peptide scanning analysis and competition experiments, we defined the sequences TIDLRY located on EC3 and KVFRVDAETGDVFAI on EC1 as the binding domain of BV6 and Cad 5, respectively. Overall, these results support the concept that VE-cadherin plays a relevant role on human endothelial cell properties. Antibodies directed to the extracellular domains EC1 but also EC3-EC4 affect VE-cadherin adhesion and clustering and alter endothelial cell permeability, apoptosis, and vascular structure formation.
Assuntos
Anticorpos Monoclonais/farmacologia , Caderinas/imunologia , Permeabilidade Capilar/efeitos dos fármacos , Endotélio Vascular/citologia , Anticorpos Monoclonais/metabolismo , Apoptose/efeitos dos fármacos , Sítios de Ligação , Caderinas/química , Adesão Celular/efeitos dos fármacos , Dimerização , Endotélio Vascular/metabolismo , Endotélio Vascular/ultraestrutura , Mapeamento de Epitopos , Humanos , Junções Intercelulares/efeitos dos fármacos , Neovascularização Fisiológica/efeitos dos fármacos , Estrutura Terciária de Proteína , Veias UmbilicaisRESUMO
Antiangiogenic therapy can enhance radiation-induced tumor growth inhibition. However, the effects of combined antiangiogenic and radiation therapy on long-term tumor control and normal tissue response have not been reported. We treated mice bearing two different human tumor xenografts with anti-vascular endothelial growth factor receptor-2 antibody (DC101) and five dose fractions of local radiation and followed them for at least 6 months. DC101 significantly decreased the dose of radiation necessary to control 50% of tumors locally. The decrease was 1.7- and 1.3-fold for the moderately radiosensitive small cell lung carcinoma 54A and the highly radioresistant glioblastoma multiforme U87, respectively. In contrast to tumors, no increase in skin radiation reaction by the antibody was detected. Surprisingly, 44% of mice bearing 54A tumor developed clear ascites after DC101 treatment at its highest dose; this was fatal to 20% of mice. This adverse effect was seen only in mice that received whole-body irradiation 1 day before tumor implantation. The encouraging results on two human tumor xenografts suggest that vascular endothelial growth factor receptor-2 blockade merits further investigation to assess its potential as an enhancer of radiation therapy in the clinic.
Assuntos
Inibidores da Angiogênese/farmacologia , Anticorpos Monoclonais/farmacologia , Carcinoma de Células Pequenas/terapia , Glioblastoma/terapia , Neoplasias Pulmonares/terapia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/imunologia , Inibidores da Angiogênese/toxicidade , Animais , Anticorpos Monoclonais/toxicidade , Peso Corporal/imunologia , Peso Corporal/efeitos da radiação , Carcinoma de Células Pequenas/irrigação sanguínea , Carcinoma de Células Pequenas/radioterapia , Divisão Celular/imunologia , Divisão Celular/efeitos da radiação , Terapia Combinada , Fracionamento da Dose de Radiação , Relação Dose-Resposta Imunológica , Relação Dose-Resposta à Radiação , Glioblastoma/irrigação sanguínea , Glioblastoma/radioterapia , Humanos , Enteropatias/etiologia , Enteropatias/imunologia , Neoplasias Pulmonares/irrigação sanguínea , Neoplasias Pulmonares/radioterapia , Masculino , Camundongos , Camundongos Nus , Oxigênio/metabolismo , Tolerância a Radiação/imunologia , Tolerância a Radiação/fisiologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Pele/efeitos da radiação , Irradiação Corporal Total/efeitos adversos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Murine vascular endothelial cadherin-2 (VE-cad-2) is a cellular adhesion molecule that is distinct from vascular endothelial cadherin 1 (VE-cad-1) in that it does not interact with catenins and does not appear to affect cell migration or growth. In this study, we have cloned a full-length cDNA of the human homolog of VE-cad-2 and used it to map the chromosomal locus of the VE-cad-2 gene. Human VE-cad-2 maps to Chromosome (Chr) 5q31. The cDNA of human VE-cad-2 is highly homologous to mouse VE-cad-2, except for a C-terminal tail. The genomic structure of VE-cad-2 is strikingly similar to that reported for a large family of neuronal protocadherin genes mapped to Chr 5q, yet the amino acid sequences between VE-cad-2 and the protocadherins are substantially divergent. The promoter of human VE-cad-2 contains two TATA boxes and transcription initiates from a single site 3' to these elements. Similar to mouse VE-cad-2, the human gene is expressed primarily in highly vascularized tissues.
Assuntos
Caderinas/genética , Caderinas/metabolismo , Cromossomos Humanos Par 5 , Endotélio Vascular/fisiologia , Regiões 5' não Traduzidas , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Éxons , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Protocaderinas , TATA BoxRESUMO
The kinase domain receptor (KDR) of vascular endothelial growth factor (VEGF) is the main human receptor responsible for the angiogenic activity of VEGF. The extracellular region of KDR is comprised of seven immunoglobulin-like domains, of which the first three have been shown to be required for ligand binding. We have previously described antibodies directed against the extracellular region of KDR, including MAB383 and MAB664, which were shown to block the binding of VEGF to the receptor and to inhibit both VEGF-induced mitogenesis of human endothelial cells in vitro and tumor growth in vivo. Here we generated a series of KDR deletion mutants consisting of truncated extracellular regions and mapped out the domain(s) responsible for binding to VEGF and the neutralizing anti-KDR antibodies. All neutralizing antibodies were found to require domain 3 for efficient binding. Alanine-scanning mutagenesis of domain 3 identified two different sets of five residues, Ile(256), Asp(257), Glu(261), Leu(313), and Thr(315) and Tyr(262), Pro(263), Ser(264), Ser(265), and Lys(266), that were critical for binding to MAB383 and MAB664, respectively. Combination of alanine mutations affecting both MAB383 and MAB664 binding resulted in a variant that also lost binding to VEGF. These results suggest that the residues within this region of domain 3 are critical for VEGF binding. Our studies provide a basis for the mechanism of action of our anti-KDR antibodies and establish a functional foundation for the development of other classes of antagonists to the receptor.
Assuntos
Anticorpos/imunologia , Fatores de Crescimento Endotelial/metabolismo , Linfocinas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Alanina/metabolismo , Sequência de Aminoácidos , Bacteriófagos/genética , Sequência de Bases , Sítios de Ligação , Primers do DNA , Mapeamento de Epitopos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Testes de Neutralização , Ligação Proteica , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento/imunologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Various conventional chemotherapeutic drugs can block angiogenesis or even kill activated, dividing endothelial cells. Such effects may contribute to the antitumor efficacy of chemotherapy in vivo and may delay or prevent the acquisition of drug-resistance by cancer cells. We have implemented a treatment regimen that augments the potential antivascular effects of chemotherapy, that is devoid of obvious toxic side effects, and that obstructs the development of drug resistance by tumor cells. Xenografts of 2 independent neuroblastoma cell lines were subjected to either continuous treatment with low doses of vinblastine, a monoclonal neutralizing antibody (DC101) targeting the flk-1/KDR (type 2) receptor for VEGF, or both agents together. The rationale for this combination was that any antivascular effects of the low-dose chemotherapy would be selectively enhanced in cells of newly formed vessels when survival signals mediated by VEGF are blocked. Both DC101 and low-dose vinblastine treatment individually resulted in significant but transient xenograft regression, diminished tumor vascularity, and direct inhibition of angiogenesis. Remarkably, the combination therapy resulted in full and sustained regressions of large established tumors, without an ensuing increase in host toxicity or any signs of acquired drug resistance during the course of treatment, which lasted for >6 months. This article may have been published online in advance of the print edition. The date of publication is available from the JCI website, http://www.jci.org.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Fitogênicos/uso terapêutico , Neuroblastoma/tratamento farmacológico , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Vimblastina/uso terapêutico , Animais , Anticorpos Monoclonais/efeitos adversos , Antineoplásicos Fitogênicos/efeitos adversos , Células Cultivadas , Terapia Combinada , Relação Dose-Resposta a Droga , Fluorescência , Humanos , Camundongos , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica , Neuroblastoma/irrigação sanguínea , Neuroblastoma/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Células Tumorais Cultivadas , Vimblastina/efeitos adversosRESUMO
Vascular endothelial-cadherin (VE-cad) is an endothelial cell-specific adhesion molecule that is crucial for proper assembly of vascular tubes. Here we show that a monoclonal antibody (BV13) directed to the extracellular region of VE-cad inhibits formation of adherens junctions and capillary-like structures by endothelial cells and blocks angiogenesis in the mouse cornea and in Matrigel plugs in vivo. Systemic administration of BV13 markedly decreases the growth of s.c. Lewis lung or human A431 epidermoid tumors and strongly suppresses the growth of Lewis lung metastases. These data demonstrate that VE-cad is essential for postnatal angiogenesis and thus validate VE-cad as a novel target for antiangiogenesis agents.
Assuntos
Anticorpos Monoclonais/farmacologia , Caderinas/imunologia , Divisão Celular/efeitos dos fármacos , Metástase Neoplásica , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Animais , Antígenos CD , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Adesão Celular , Colágeno/metabolismo , Córnea/efeitos dos fármacos , Relação Dose-Resposta a Droga , Combinação de Medicamentos , Endotélio/metabolismo , Humanos , Imunoglobulina G/metabolismo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Laminina/metabolismo , Neoplasias Pulmonares/tratamento farmacológico , Camundongos , Camundongos Nus , Transplante de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteoglicanas/metabolismo , Fatores de Tempo , Células Tumorais CultivadasRESUMO
Bispecific antibody (BsAb) technology has been successfully used as a means to construct novel antibody (Ab) molecules with increased avidity for binding, by combining two Ab or their fragments directed against different epitopes within the same antigen. Using two single chain antibodies (scFv) isolated from a phage display library, we have constructed a bispecific diabody directed against two different epitopes on the extracellular domain (ECD) of human vascular endothelial growth factor receptor 2 (VEGFR2), the kinase-insert domain-containing receptor (KDR). Neither of the parent scFv blocks KDR/VEGF interactions or inhibits VEGF-induced receptor activation. The diabody binds to KDR with an affinity that is 1.5- to 3-fold higher than its parent scFv, mainly due to a much slower dissociation rate (k(off)), which is approximately 17- to 26-fold slower than that of the individual scFv. In addition, the diabody binds simultaneously to, and thus cross-links, the two epitopes on the receptor(s). It is rather unexpected that the diabody effectively blocked KDR/VEGF interactions, and inhibited both VEGF-induced activation of the receptor and mitogenesis of human endothelial cells. Taken together, our results suggest that the diabody is most likely to exert its effect through steric hindrance and/or causing major conformational changes of the receptor. This is the first report on the construction of a bispecific diabody with acquired novel antagonistic activity.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Biespecíficos/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores de Fatores de Crescimento/imunologia , Afinidade de Anticorpos , Especificidade de Anticorpos , Linhagem Celular , Epitopos/química , Escherichia coli/genética , Humanos , Técnicas Imunológicas , Testes de Neutralização , Estrutura Terciária de Proteína , Receptores Proteína Tirosina Quinases/química , Receptores de Fatores de Crescimento/química , Receptores de Fatores de Crescimento do Endotélio VascularRESUMO
Tumor angiogenesis is mediated by tumor-secreted angiogenic growth factors that interact with their surface receptors expressed on endothelial cells. Vascular endothelial growth factor (VEGF) and its receptor [fetal liver kinase 1 (Flk-1)/kinase insert domain-containing receptor] play an important role in vascular permeability and tumor angiogenesis. Previously, we reported on the development of anti-Flk-1 and antikinase insert domain-containing receptor monoclonal antibodies (mAbs) that potently inhibit VEGF binding and receptor signaling. Here, we report the effect of anti-Flk-1 mAb (DC101) on angiogenesis and tumor growth. Angiogenesis in vivo was examined using a growth factor supplemented (basic fibroblast growth factor + VEGF) Matrigel plug and an alginate-encapsulated tumor cell (Lewis lung) assay in C57BL/6 mice. Systemic administration of DC101 every 3 days markedly reduced neovascularization of Matrigel plugs and tumor-containing alginate beads in a dose-dependent fashion. Histological analysis of Matrigel plugs showed reduced numbers of endothelial cells and vessel structures. Several mouse tumors and human tumor xenografts in athymic mice were used to examine the effect of anti-Flk-1 mAb treatment on tumor angiogenesis and growth. Anti-Flk-1 mAb treatment significantly suppressed the growth of primary murine Lewis lung, 4T1 mammary, and B16 melanoma tumors and growth of Lewis lung metastases. DC101 also completely inhibited the growth of established epidermoid, glioblastoma, pancreatic, and renal human tumor xenografts. Histological examination of anti-Flk-1 mAb-treated tumors showed evidence of decreased microvessel density, tumor cell apoptosis, decreased tumor cell proliferation, and extensive tumor necrosis. These findings support the conclusion that anti-Flk-1 mAb treatment inhibits tumor growth by suppression of tumor-induced neovascularization and demonstrate the potential for therapeutic application of anti-VEGF receptor antibody in the treatment of angiogenesis-dependent tumors.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Neoplasias Experimentais/terapia , Neovascularização Patológica/prevenção & controle , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Animais , Apoptose , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Nus , Necrose , Transplante de Neoplasias , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/patologia , Receptores de Fatores de Crescimento do Endotélio Vascular , Transplante HeterólogoRESUMO
We have cloned a gene (HSE1) from a human placental cDNA library that encodes a novel protein exhibiting heparanase activity. The cDNA was identified through peptide sequences derived from purified heparanase isolated from human SK-HEP-1 hepatoma cells. HSE1 contains an open reading frame encoding a predicted polypeptide of 543 amino acids and possesses a putative signal sequence at its amino terminus. Northern blot analysis suggested strong expression of HSE1 in placenta and spleen. Transient transfection of HSE1 in COS7 cells resulted in the expression of a protein with an apparent molecular mass of 67-72 kDa. HSE1 protein was detectable in conditioned media but was also associated with the membrane fraction following cell lysis. The HSE1 gene product was shown to exhibit heparanase activity by specifically cleaving a labeled heparan sulfate substrate in a similar manner as purified native protein.
Assuntos
Expressão Gênica , Glucuronidase , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Membrana Celular/enzimologia , Clonagem Molecular , Etiquetas de Sequências Expressas , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/isolamento & purificação , Glicosídeo Hidrolases/metabolismo , Glicosilação , Heparitina Sulfato/metabolismo , Humanos , Dados de Sequência Molecular , Peso Molecular , Fases de Leitura Aberta/genética , Especificidade de Órgãos , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Fragmentos de Peptídeos/metabolismo , Placenta/enzimologia , Placenta/metabolismo , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Solubilidade , Baço/enzimologia , Baço/metabolismo , TransfecçãoRESUMO
The kinase insert domain-containing receptor (KDR) is the human vascular endothelial growth factor (VEGF) receptor responsible for the mitogenic and angiogenic effects of VEGF. There is much experimental evidence to suggest that the VEGF/KDR pathway plays an important role in tumor angiogenesis, a process essential for tumor growth and metastasis. Here we produced a chimeric anti-KDR antibody (IgG1), c-p1C11, from a single chain (scFv) antibody isolated from a phage display library. C-p1C11 binds specifically to the extracellular domain of soluble as well as cell-surface expressed KDR. It effectively blocks VEGF-KDR interaction and inhibits VEGF-stimulated activation of KDR and MAP kinases p44/p42 of human endothelial cells. Furthermore, c-p1C11 efficiently neutralizes VEGF-induced mitogenesis of human endothelial cells. Our results suggest that antibodies against KDR have potential clinical applications in the treatment of cancer and other diseases where pathological angiogenesis is involved.