RESUMO
Bv8, also known as prokineticin 2, has been characterized as an important mediator of myeloid cell mobilization and myeloid cell-dependent tumor angiogenesis. Bv8 expression is dramatically enhanced by G-CSF, both in vitro and in vivo. The mechanisms involved in such up-regulation remain unknown. Using pharmacological inhibitors that interfere with multiple signaling pathways known to be activated by G-CSF, we show that signal transducer and activator of transcription 3 (Stat3) activation is required for Bv8 up-regulation in mouse bone marrow cells, whereas other Stat family members and extracellular signal-regulated kinase (ERK) activation are not involved. We further identified CD11b(+) Gr1(+) myeloid cells as the primary cell population in which Stat3 signaling is activated by G-CSF. Bv8 expression induced by G-CSF was also significantly reduced by siRNA-mediated Stat3 knockdown. Moreover, chromatin immunoprecipitation studies indicate that G-CSF significantly induces binding of phospho-Stat3 to the Bv8 promoter, which was abolished by pretreatment with the Stat3 inhibitor WP1066. Luciferase assay confirmed that the phospho-Stat3 binding site is a functional enhancer of the Bv8 promoter. The key role of Stat3 signaling in regulating G-CSF-induced Bv8 expression was further confirmed by in vivo studies. We show that the regulation of Bv8 expression in human bone marrow cells is also Stat3 signaling-dependent. Stat3 is recognized as a key regulator of inflammation-dependent tumorigenesis. We propose that such a role of Stat3 reflects at least in part its ability to regulate Bv8 expression.
Assuntos
Células da Medula Óssea/metabolismo , Antígeno CD11b , Hormônios Gastrointestinais/biossíntese , Regulação da Expressão Gênica/fisiologia , Fator Estimulador de Colônias de Granulócitos/farmacologia , Neuropeptídeos/biossíntese , Receptores de Superfície Celular , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Células da Medula Óssea/citologia , Células Cultivadas , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Hormônios Gastrointestinais/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Neuropeptídeos/genética , Regiões Promotoras Genéticas/fisiologia , Piridinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/genética , Transdução de Sinais/fisiologia , Tirfostinas/farmacologiaRESUMO
Upper hinge is vulnerable to radical attacks that result in breakage of the heavy-light chain linkage and cleavage of the hinge of an IgG1. To further explore mechanisms responsible for the radical induced hinge degradation, nine mutants were designed to determine the roles that the upper hinge Asp and His play in the radical reactions. The observation that none of these substitutions could inhibit the breakage of the heavy-light chain linkage suggests that the breakage may result from electron transfer from Cys(231) directly to the heavy-light chain linkage upon radical attacks, and implies a pathway separate from His(229)-mediated hinge cleavage. On the other hand, the substitution of His(229) with Tyr showed promising advantages over the native antibody and other substitutions in improving the stability and function of the IgG1. This substitution inhibited the hinge cleavage by 98% and suggests that the redox active nature of Tyr did not enable it to replicate the ability of His to facilitate radical induced degradation. We propose that the lower redox potential of Tyr, a residue that may be the ultimate sink for oxidizing equivalents in proteins, is responsible for the inhibition. More importantly, the substitution increased the antibody's binding to FcγRIII receptors by 2-3-fold, and improved ADCC activity by 2-fold, while maintaining a similar pharmacokinetic profile with respect to the wild type. Implications of these observations for antibody engineering and development are discussed.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Engenharia de Proteínas/métodos , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Cricetinae , Cricetulus , Estudos de Viabilidade , Humanos , Radical Hidroxila/farmacologia , Imunoglobulina G/genética , Cadeias Pesadas de Imunoglobulinas/química , Cadeias Leves de Imunoglobulina/química , Dados de Sequência Molecular , Mutação , Estabilidade Proteica , Proteólise/efeitos dos fármacosRESUMO
Antibodies to cell-surface antigens trigger activatory Fcγ receptor (FcγR)-mediated retrograde signals in leukocytes to control immune effector functions. Here, we uncover an FcγR mechanism that drives antibody-dependent forward signaling in target cells. Agonistic antibodies to death receptor 5 (DR5) induce cancer-cell apoptosis and are in clinical trials; however, their mechanism of action in vivo is not fully defined. Interaction of the DR5-agonistic antibody drozitumab with leukocyte FcγRs promoted DR5-mediated tumor-cell apoptosis. Whereas the anti-CD20 antibody rituximab required activatory FcγRs for tumoricidal function, drozitumab was effective in the context of either activatory or inhibitory FcγRs. A CD40-agonistic antibody required similar FcγR interactions to stimulate nuclear factor-κB activity in B cells. Thus, FcγRs can drive antibody-mediated receptor signaling in target cells.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias/metabolismo , Receptores de IgG/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Transdução de Sinais/imunologia , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Anticorpos Monoclonais Humanizados , Apoptose/imunologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Antígenos CD40/agonistas , Antígenos CD40/imunologia , Linhagem Celular Tumoral , Feminino , Células HCT116 , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Imunoglobulina G/farmacologia , Células Matadoras Naturais/imunologia , Leucócitos/imunologia , Leucócitos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Mutação/imunologia , Células Mieloides/imunologia , NF-kappa B/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Polimorfismo de Nucleotídeo Único/genética , Polimorfismo de Nucleotídeo Único/imunologia , Ligação Proteica/genética , Ligação Proteica/imunologia , Agregação de Receptores/imunologia , Receptores de IgG/genética , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/agonistas , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The enhancement of immune effector functions has been proposed as a potential strategy for increasing the efficacy of therapeutic antibodies. Here, we show that removing fucose from trastuzumab (Herceptin) increased its binding to FcgammaRIIIa, enhanced antibody-dependent cell-mediated cytotoxicity, and more than doubled the median progression-free survival when compared with conventional trastuzumab in treating preclinical models of HER2-amplified breast cancer. Our results show that afucosylated trastuzumab has superior efficacy in treating in vivo models of HER2-amplified breast cancer and support the development of effector function-enhanced antibodies for solid tumor therapy.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/enzimologia , Receptor ErbB-2/metabolismo , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Anticorpos Monoclonais Humanizados , Citotoxicidade Celular Dependente de Anticorpos/efeitos dos fármacos , Antineoplásicos/farmacologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Fucose/química , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Camundongos Transgênicos , Receptores de IgG/biossíntese , Receptores de IgG/genética , Receptores de IgG/metabolismo , TrastuzumabRESUMO
Activated fibroblasts are thought to play important roles in the progression of many solid tumors, but little is known about the mechanisms responsible for the recruitment of fibroblasts in tumors. Using several methods, we identified platelet-derived growth factor A (PDGFA) as the major fibroblast chemoattractant and mitogen from conditioned medium generated by the Calu-6 lung carcinoma cell line. In addition, we showed that Calu-6 tumors express significant levels of PDGFC, and that the levels of expression of these two PDGFRalpha ligands correlate strongly with the degree of stromal fibroblast infiltration into the tumor mass. The most intense expression of PDGFRalpha was observed in fibroblasts in the tumor outer rim. We subsequently showed that disrupting PDGFRalpha-mediated signaling results in significant inhibition of tumor growth in vivo. Furthermore, analysis of a compendium of microarray data revealed significant expression of PDGFA, PDGFC, and PDGFRalpha in human lung tumors. We propose that therapies targeting this stromal cell type may be effective in treating certain types of solid tumors.
Assuntos
Neoplasias Pulmonares/fisiopatologia , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Células Estromais/patologia , Células 3T3 , Animais , Anticorpos , Divisão Celular , Linhagem Celular Tumoral , Meios de Cultivo Condicionados , Primers do DNA , Regulação Neoplásica da Expressão Gênica , Humanos , Pulmão/citologia , Pulmão/fisiologia , Neoplasias Pulmonares/patologia , Linfocinas/genética , Camundongos , Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/isolamento & purificação , Valores de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de SinaisRESUMO
Vascular endothelial growth factor-A (VEGF-A) and its 2 transmembrane tyrosine-kinase receptors, VEGFR-1 and VEGFR-2, constitute a ligand-receptor signaling system that is crucial for developmental angiogenesis. VEGF-B and placental growth factor (PlGF) activate VEGFR-1 selectively, however, mice lacking either ligand display only minor developmental defects. We hypothesized that the relative contributions of VEGF-B and PlGF to VEGFR-1 signaling may be masked in the presence of VEGF-A, which is abundantly expressed during postnatal development. To test this hypothesis, neonatal or adult mice were treated with a monoclonal antibody (G6-23-IgG) blocking murine VEGF-A or a soluble VEGFR-1 receptor IgG chimeric construct [mFlt(1-3)-IgG], which neutralizes VEGF-A, VEGF-B, and PlGF. Both compounds attenuated growth and survival of neonatal mice to similar extents and the pathophysiologic alterations, including a reduction in organ size and vascularization, changes in gene expression, and hematologic end points, were essentially indistinguishable. In adult mice, we observed only minor changes in response to treatment, which were similar between both anti-VEGF compounds. In conclusion, our findings suggest that PlGF and VEGF-B do not compensate during conditions of VEGF-A blockade, suggesting a minor role for compensatory VEGFR-1 signaling during postnatal development and vascular homeostasis in adults. The absence of compensatory VEGFR-1 signaling by VEGF-B and PlGF may have important implications for the development of anticancer strategies targeting the VEGF ligand/receptor system.
Assuntos
Neoplasias Experimentais/terapia , Neovascularização Patológica , Proteínas da Gravidez/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator B de Crescimento do Endotélio Vascular/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Camundongos , Camundongos Mutantes , Camundongos Nus , Neoplasias Experimentais/irrigação sanguínea , Neoplasias Experimentais/metabolismo , Fator de Crescimento Placentário , Proteínas da Gravidez/antagonistas & inibidores , Proteínas da Gravidez/imunologia , Taxa de Sobrevida , Fator A de Crescimento do Endotélio Vascular/imunologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator B de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator B de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/imunologia , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
Some Abs are more efficacious after being cross-linked to form dimers or multimers, presumably as a result of binding to and clustering more surface target to either amplify or diversify cellular signaling. To improve the therapeutic potency of these types of Abs, we designed and generated Abs that express tandem Fab repeats with the aim of mimicking cross-linked Abs. The versatile design of the system enables the creation of a series of multivalent human IgG Ab forms including tetravalent IgG1, tetravalent F(ab')2, and linear Fab multimers with either three or four consecutively linked Fabs. The multimerized Abs target the cell surface receptors HER2, death receptor 5, and CD20, and are more efficacious than their parent mAbs in triggering antitumor cellular responses, indicating they could be useful both as reagents for study as well as novel therapeutics.
Assuntos
Anticorpos Monoclonais/química , Animais , Anticorpos Monoclonais/biossíntese , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Afinidade de Anticorpos , Antígenos CD20/imunologia , Antígenos CD20/metabolismo , Apoptose/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Espaço Extracelular/imunologia , Espaço Extracelular/metabolismo , Meia-Vida , Humanos , Fragmentos Fab das Imunoglobulinas/biossíntese , Fragmentos Fab das Imunoglobulinas/química , Fragmentos Fab das Imunoglobulinas/metabolismo , Fragmentos de Imunoglobulinas/biossíntese , Fragmentos de Imunoglobulinas/química , Fragmentos de Imunoglobulinas/metabolismo , Imunoglobulina G/biossíntese , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Masculino , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Receptor ErbB-2/imunologia , Receptor ErbB-2/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF , Receptores do Fator de Necrose Tumoral/imunologia , Receptores do Fator de Necrose Tumoral/metabolismo , Frações Subcelulares/química , Frações Subcelulares/imunologia , Frações Subcelulares/metabolismo , Células Tumorais CultivadasRESUMO
OBJECTIVES: To evaluate the toxicity and activity of thalidomide in patients with advanced metastatic renal cell cancer and to measure changes of one angiogenic factor, vascular endothelial growth factor (VEGF)165, with therapy. PATIENTS AND METHODS: 29 patients were enrolled on a study of thalidomide using an intra-patient dose escalation schedule. Patients began thalidomide at 400 mg/d and escalated as tolerated to 1200 mg/d by day 54. Fifty-nine per cent of patients had had previous therapy with IL-2 and 52% were performance status 2 or 3. Systemic plasma VEGF165 levels were measured by dual monoclonal ELISA in 8 patients. RESULTS: 24 patients were evaluable for response with one partial response of 11 months duration of a patient with hepatic and pulmonary metastases (4%), one minor response, and 2 patients stable for over 6 months. Somnolence and constipation were prominent toxicities and most patients could not tolerate the 1200 mg/day dose level. Systemic plasma VEGF165 levels did not change with therapy. CONCLUSION: These results are consistent with a low level of activity of thalidomide in renal cell carcinoma. Administration of doses over 800 mg/day was difficult to achieve in this patient population, however lower doses were practical. The dose-response relationship, if any, of thalidomide for renal cell carcinoma is unclear.
Assuntos
Carcinoma de Células Renais/tratamento farmacológico , Neoplasias Renais/tratamento farmacológico , Talidomida/efeitos adversos , Talidomida/uso terapêutico , Adulto , Idoso , Carcinoma de Células Renais/sangue , Carcinoma de Células Renais/secundário , Constipação Intestinal/induzido quimicamente , Distúrbios do Sono por Sonolência Excessiva/induzido quimicamente , Fatores de Crescimento Endotelial/sangue , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/sangue , Neoplasias Renais/sangue , Neoplasias Renais/secundário , Linfocinas/sangue , Masculino , Pessoa de Meia-Idade , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular endothelial growth factor (VEGF) is a principal regulator of blood vessel formation and haematopoiesis, but the mechanisms by which VEGF differentially regulates these processes have been elusive. Here we describe a regulatory loop by which VEGF controls survival of haematopoietic stem cells (HSCs). We observed a reduction in survival, colony formation and in vivo repopulation rates of HSCs after ablation of the VEGF gene in mice. Intracellularly acting small-molecule inhibitors of VEGF receptor (VEGFR) tyrosine kinase dramatically reduced colony formation of HSCs, thus mimicking deletion of the VEGF gene. However, blocking VEGF by administering a soluble VEGFR-1, which acts extracellularly, induced only minor effects. These findings support the involvement in HSC survival of a VEGF-dependent internal autocrine loop mechanism (that is, the mechanism is resistant to inhibitors that fail to penetrate the intracellular compartment). Not only ligands selective for VEGF and VEGFR-2 but also VEGFR-1 agonists rescued survival and repopulation of VEGF-deficient HSCs, revealing a function for VEGFR-1 signalling during haematopoiesis.
Assuntos
Comunicação Autócrina , Fatores de Crescimento Endotelial/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Linfocinas/metabolismo , Animais , Comunicação Autócrina/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Clonais/citologia , Células Clonais/efeitos dos fármacos , Células Clonais/metabolismo , Fatores de Crescimento Endotelial/antagonistas & inibidores , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/citologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Citometria de Fluxo , Deleção de Genes , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Humanos , Linfocinas/antagonistas & inibidores , Linfocinas/genética , Camundongos , Camundongos Knockout , Comunicação Parácrina , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Proteína Tirosina Quinases/farmacologia , Receptores de Fatores de Crescimento/agonistas , Receptores de Fatores de Crescimento/antagonistas & inibidores , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solubilidade , Transdução Genética , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Many research and clinical applications require large quantities of full-length antibodies with long circulating half-lives, and production of these complex multi-subunit proteins has in the past been restricted to eukaryotic hosts. In this report, we demonstrate that efficient secretion of heavy and light chains in a favorable ratio leads to the high-level expression and assembly of full-length IgGs in the Escherichia coli periplasm. The technology described offers a rapid, generally applicable and potentially inexpensive method for the production of full-length therapeutic antibodies, as verified by the expression of several humanized IgGs. One E. coli-derived antibody in particular, anti-tissue factor IgG1, has been thoroughly evaluated and has all of the expected properties of an aglycosylated antibody, including tight binding to antigen and the neonatal receptor. As predicted, the protein lacks binding to C1q and the FcgammaRI receptor, making it an ideal candidate for research purposes and therapeutic indications where effector functions are either not required or are actually detrimental. In addition, a limited chimpanzee study suggests that the E. coli-derived IgG1 retains the long circulating half-life of mammalian cell-derived antibodies.