RESUMO
It has been recently reported that treatment with an anti-placenta growth factor (PlGF) antibody inhibits metastasis and primary tumor growth. Here we show that, although anti-PlGF treatment inhibited wound healing, extravasation of B16F10 cells, and growth of a tumor engineered to overexpress the PlGF receptor (VEGFR-1), neutralization of PlGF using four novel blocking antibodies had no significant effect on tumor angiogenesis in 15 models. Also, genetic ablation of the tyrosine kinase domain of VEGFR-1 in the host did not result in growth inhibition of the anti-VEGF-A sensitive or resistant tumors tested. Furthermore, combination of anti-PlGF with anti-VEGF-A antibodies did not result in greater antitumor efficacy than anti-VEGF-A monotherapy. In conclusion, our data argue against an important role of PlGF during primary tumor growth in most models and suggest that clinical evaluation of anti-PlGF antibodies may be challenging.
Assuntos
Neoplasias/irrigação sanguínea , Neovascularização Patológica , Proteínas da Gravidez/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Linhagem Celular Tumoral , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Placentário , Proteínas da Gravidez/antagonistas & inibidores , Fatores de Crescimento do Endotélio VascularRESUMO
Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Terapia de Alvo Molecular , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Proteínas Tirosina Fosfatases Classe 2 Semelhantes a Receptores/metabolismo , Trombospondinas/metabolismo , Animais , Anticorpos/imunologia , Anticorpos/farmacologia , Anticorpos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Progressão da Doença , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Mucosa Intestinal/metabolismo , Intestinos/citologia , Intestinos/efeitos dos fármacos , Intestinos/patologia , Masculino , Camundongos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Trombospondinas/antagonistas & inibidores , Trombospondinas/imunologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The proto-oncogenes ETV1, ETV4 and ETV5 encode transcription factors in the E26 transformation-specific (ETS) family, which includes the most frequently rearranged and overexpressed genes in prostate cancer. Despite being critical regulators of development, little is known about their post-translational regulation. Here we identify the ubiquitin ligase COP1 (also known as RFWD2) as a tumour suppressor that negatively regulates ETV1, ETV4 and ETV5. ETV1, which is mutated in prostate cancer more often, was degraded after being ubiquitinated by COP1. Truncated ETV1 encoded by prostate cancer translocation TMPRSS2:ETV1 lacks the critical COP1 binding motifs and was 50-fold more stable than wild-type ETV1. Almost all patient translocations render ETV1 insensitive to COP1, implying that this confers a selective advantage to prostate epithelial cells. Indeed, COP1 deficiency in mouse prostate elevated ETV1 and produced increased cell proliferation, hyperplasia, and early prostate intraepithelial neoplasia. Combined loss of COP1 and PTEN enhanced the invasiveness of mouse prostate adenocarcinomas. Finally, rare human prostate cancer samples showed hemizygous loss of the COP1 gene, loss of COP1 protein, and elevated ETV1 protein while lacking a translocation event. These findings identify COP1 as a tumour suppressor whose downregulation promotes prostatic epithelial cell proliferation and tumorigenesis.
Assuntos
Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-ets/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Masculino , Camundongos , Proteínas Nucleares/deficiência , PTEN Fosfo-Hidrolase/deficiência , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ligação Proteica , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Human cytomegalovirus (HCMV) is the most common cause of congenital virus infection. Congenital HCMV infection occurs in 0.2-1% of all births, and causes birth defects and developmental abnormalities, including sensorineural hearing loss and developmental delay. Several key studies have established the guinea pig as a tractable model for the study of congenital HCMV infection and have shown that polyclonal antibodies can be protective. In this study, we demonstrate that an anti-guinea pig CMV (GPCMV) glycoprotein H/glycoprotein L neutralizing monoclonal antibody protects against fetal infection and loss in the guinea pig. Furthermore, we have delineated the kinetics of GPCMV congenital infection, from maternal infection (salivary glands, seroconversion, placenta) to fetal infection (fetus and amniotic fluid). Our studies support the hypothesis that a neutralizing monoclonal antibody targeting an envelope GPCMV glycoprotein can protect the fetus from infection and may shed light on the therapeutic intervention of HCMV congenital infection in humans.
Assuntos
Anticorpos Monoclonais Murinos/farmacologia , Anticorpos Neutralizantes/farmacologia , Anticorpos Antivirais/farmacologia , Infecções por Citomegalovirus/congênito , Infecções por Citomegalovirus/tratamento farmacológico , Citomegalovirus/imunologia , Animais , Anticorpos Monoclonais Murinos/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por Citomegalovirus/imunologia , Infecções por Citomegalovirus/patologia , Modelos Animais de Doenças , Cobaias , Células HEK293 , HumanosRESUMO
Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-4(-/-) resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-alpha in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs.
Assuntos
Homeostase/imunologia , Macrófagos Peritoneais/imunologia , Proteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Anticorpos/imunologia , Formação de Anticorpos/imunologia , Apoptose/imunologia , Adesão Celular , Contagem de Células , Linhagem Celular , Eritrócitos/imunologia , Humanos , Hipersensibilidade Tardia/imunologia , Macrófagos Peritoneais/citologia , Proteínas de Membrana/deficiência , Camundongos , Fagocitose/imunologia , Transporte Proteico , Receptores de Complemento/imunologia , Ovinos , Baço/citologia , Baço/imunologia , Fator de Necrose Tumoral alfa/biossínteseRESUMO
Haploinsufficiency of Dll4, a vascular-specific Notch ligand, has shown that it is essential for embryonic vascular development and arteriogenesis. Mechanistically, it is unclear how the Dll4-mediated Notch pathway contributes to complex vascular processes that demand meticulous coordination of multiple signalling pathways. Here we show that Dll4-mediated Notch signalling has a unique role in regulating endothelial cell proliferation and differentiation. Neutralizing Dll4 with a Dll4-selective antibody rendered endothelial cells hyperproliferative, and caused defective cell fate specification or differentiation both in vitro and in vivo. In addition, blocking Dll4 inhibited tumour growth in several tumour models. Remarkably, antibodies against Dll4 and antibodies against vascular endothelial growth factor (VEGF) had paradoxically distinct effects on tumour vasculature. Our data also indicate that Dll4-mediated Notch signalling is crucial during active vascularization, but less important for normal vessel maintenance. Furthermore, unlike blocking Notch signalling globally, neutralizing Dll4 had no discernable impact on intestinal goblet cell differentiation, supporting the idea that Dll4-mediated Notch signalling is largely restricted to the vascular compartment. Therefore, targeting Dll4 might represent a broadly efficacious and well-tolerated approach for the treatment of solid tumours.
Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Neoplasias/irrigação sanguínea , Neoplasias/patologia , Neovascularização Patológica , Transdução de Sinais , Animais , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Endotélio Vascular/citologia , Homeostase , Humanos , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Receptores Notch/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Luminal A (hormone receptor-positive) breast cancer constitutes 70% of total breast cancer patients. In an attempt to develop a targeted therapeutic for this cancer indication, we have identified and characterized Glial cell line-Derived Neurotrophic Factor (GDNF) Family Receptor Alpha 1 (GFRA1) antibody-drug conjugates (ADC) using a cleavable valine-citrulline-MMAE (vcMMAE) linker-payload. RNAseq and IHC analysis confirmed the abundant expression of GFRA1 in luminal A breast cancer tissues, whereas minimal or no expression was observed in most normal tissues. Anti-GFRA-vcMMAE ADC internalized to the lysosomes and exhibited target-dependent killing of GFRA1-expressing cells both in vitro and in vivo The ADCs using humanized anti-GFRA1 antibodies displayed robust therapeutic activity in clinically relevant cell line-derived (MCF7 and KPL-1) tumor xenograft models. The lead anti-GFRA1 ADC cross-reacts with rodent and cynomolgus monkey GFRA1 antigen and showed optimal pharmacokinetic properties in both species. These properties subsequently enabled a target-dependent toxicity study in rats. Anti-GFRA1 ADC is well tolerated in rats, as seen with other vcMMAE linker-payload based ADCs. Overall, these data suggest that anti-GFRA1-vcMMAE ADC may provide a targeted therapeutic opportunity for luminal A breast cancer patients. Mol Cancer Ther; 17(3); 638-49. ©2017 AACR.
Assuntos
Neoplasias da Mama/tratamento farmacológico , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/antagonistas & inibidores , Imunoconjugados/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Anticorpos/química , Anticorpos/imunologia , Anticorpos/farmacologia , Antineoplásicos/química , Antineoplásicos/farmacologia , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/imunologia , Células HEK293 , Humanos , Imunoconjugados/imunologia , Imunoconjugados/farmacocinética , Células MCF-7 , Macaca fascicularis , Camundongos Nus , Camundongos SCID , Ratos Sprague-Dawley , Receptores de Esteroides/metabolismo , Carga Tumoral/efeitos dos fármacos , Carga Tumoral/genéticaRESUMO
Bispecific antibodies offer a clinically validated platform for drug discovery. In generating functionally active bispecific antibodies, it is necessary to identify a unique parental antibody pair to merge into a single molecule. However, technologies that allow high-throughput production of bispecific immunoglobulin Gs (BsIgGs) for screening purposes are limited. Here, we describe a novel bispecific antibody format termed tethered-variable CLBsIgG (tcBsIgG) that allows robust production of intact BsIgG in a single cell line, concurrently ensuring cognate light chain pairing and preserving key antibody structural and functional properties. This technology is broadly applicable in the generation of BsIgG from a variety of antibody isotypes, including human BsIgG1, BsIgG2 and BsIgG4. The practicality of the tcBsIgG platform is demonstrated by screening BsIgGs generated from FGF21-mimetic anti-Klotho-ß agonistic antibodies in a combinatorial manner. This screen identified multiple biepitopic combinations with enhanced agonistic activity relative to the parental monoclonal antibodies, thereby demonstrating that biepitopic antibodies can acquire enhanced functionality compared to monospecific parental antibodies. By design, the tcBsIgG format is amenable to high-throughput production of large panels of bispecific antibodies and thus can facilitate the identification of rare BsIgG combinations to enable the discovery of molecules with improved biological function.
Assuntos
Anticorpos Biespecíficos/biossíntese , Anticorpos Monoclonais/biossíntese , Ensaios de Triagem em Larga Escala , Imunoglobulina G/biossíntese , Engenharia de Proteínas/métodos , Animais , Anticorpos Biespecíficos/química , Anticorpos Biespecíficos/genética , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Células CHO , Clonagem Molecular , Cricetulus , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/imunologia , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Imunoglobulina G/química , Imunoglobulina G/genética , Proteínas Klotho , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/imunologia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We conducted an expression analysis of prostate stem cell antigen (PSCA)in normal urogenital tissues, benign prostatic hyperplasia (n = 21), prostatic intraepithelial neoplasia (n = 33), and primary (n = 137) and metastatic (n = 42) prostate adenocarcinoma, using isotopic in situ hybridization on tissue microarrays. In normal prostate, we observe PSCA expression in the terminally differentiated, secretory epithelium; strong expression was also seen in normal urothelium. Forty-eight percent of primary and 64% of metastatic prostatic adenocarcinomas expressed PSCA RNA. Our studies did not confirm a positive correlation between level of PSCA RNA expression and high Gleason grade. We characterized monoclonal anti-PSCA antibodies that recognize PSCA expressed on the surface of live cells, are efficiently internalized after antigen recognition, and kill tumor cells in vitro in an antigen-specific fashion upon conjugation with maytansinoid. Unconjugated anti-PSCA antibodies demonstrated efficacy against PSCA-positive tumors by delaying progressive tumor growth in vivo. Maytansinoid-conjugated antibodies caused complete regression of established tumors in a large proportion of animals. Our results strongly suggest that maytansinoid-conjugated anti-PSCA monoclonal antibodies should be evaluated as a therapeutic modality for patients with advanced prostate cancer.
Assuntos
Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Anticorpos Monoclonais/farmacologia , Glicoproteínas de Membrana/imunologia , Proteínas de Neoplasias/imunologia , Neoplasias da Próstata/imunologia , Neoplasias da Próstata/terapia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacocinética , Antígenos de Neoplasias , Feminino , Proteínas Ligadas por GPI , Humanos , Imunização Passiva/métodos , Imunotoxinas/farmacocinética , Imunotoxinas/farmacologia , Hibridização In Situ , Masculino , Maitansina/farmacocinética , Maitansina/farmacologia , Glicoproteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Pessoa de Meia-Idade , Proteínas de Neoplasias/biossíntese , Neoplasias da Próstata/metabolismoRESUMO
Eosinophilic inflammation and Th2 cytokine production are central to the pathogenesis of asthma. Agents that target either eosinophils or single Th2 cytokines have shown benefits in subsets of biomarker-positive patients. More broadly effective treatment or disease-modifying effects may be achieved by eliminating more than one inflammatory stimulator. Here we present a strategy to concomitantly deplete Th2 T cells, eosinophils, basophils, and type-2 innate lymphoid cells (ILC2s) by generating monoclonal antibodies with enhanced effector function (19A2) that target CRTh2 present on all 4 cell types. Using human CRTh2 (hCRTh2) transgenic mice that mimic the expression pattern of hCRTh2 on innate immune cells but not Th2 cells, we demonstrate that anti-hCRTh2 antibodies specifically eliminate hCRTh2+ basophils, eosinophils, and ILC2s from lung and lymphoid organs in models of asthma and Nippostrongylus brasiliensis infection. Innate cell depletion was accompanied by a decrease of several Th2 cytokines and chemokines. hCRTh2-specific antibodies were also active on human Th2 cells in vivo in a human Th2-PBMC-SCID mouse model. We developed humanized hCRTh2-specific antibodies that potently induce antibody-dependent cell cytotoxicity (ADCC) of primary human eosinophils and basophils and replicated the in vivo depletion capacity of their murine parent. Therefore, depletion of hCRTh2+ basophils, eosinophils, ILC2, and Th2 cells with h19A2 hCRTh2-specific antibodies may be a novel and more efficacious treatment for asthma.
Assuntos
Citotoxicidade Celular Dependente de Anticorpos , Asma/terapia , Células Th2/citologia , Animais , Anticorpos Monoclonais Humanizados/imunologia , Basófilos/citologia , Citocinas , Modelos Animais de Doenças , Eosinófilos/citologia , Humanos , Imunidade Inata , Pulmão/citologia , Pulmão/imunologia , Linfócitos/citologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos SCID , Camundongos TransgênicosRESUMO
Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers.
Assuntos
Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Neoplasias do Colo/tratamento farmacológico , Imunotoxinas/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Receptores Acoplados a Proteínas G/imunologia , Animais , Antineoplásicos/imunologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/genética , Neoplasias do Colo/imunologia , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Relação Dose-Resposta a Droga , Estudos de Viabilidade , Feminino , Regulação Neoplásica da Expressão Gênica , Genes APC , Imunotoxinas/imunologia , Imunotoxinas/metabolismo , Concentração Inibidora 50 , Masculino , Camundongos Endogâmicos C57BL , Camundongos Nus , Camundongos Transgênicos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Fatores de Tempo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Multi-transmembrane proteins are especially difficult targets for antibody generation largely due to the challenge of producing a protein that maintains its native conformation in the absence of a stabilizing membrane. Here, we describe an immunization strategy that successfully resulted in the identification of monoclonal antibodies that bind specifically to extracellular epitopes of a 12 transmembrane protein, multi-drug resistant protein 4 (MRP4). These monoclonal antibodies were developed following hydrodynamic tail vein immunization with a cytomegalovirus (CMV) promoter-based plasmid expressing MRP4 cDNA and were characterized by flow cytometry. As expected, the use of the immune modulators fetal liver tyrosine kinase 3 ligand (Flt3L) and granulocyte-macrophage colony-stimulating factor positively enhanced the immune response against MRP4. Imaging studies using CMV-based plasmids expressing luciferase showed that the in vivo half-life of the target antigen was less than 48 h using CMV-based plasmids, thus necessitating frequent boosting with DNA to achieve an adequate immune response. We also describe a comparison of plasmids, which contained MRP4 cDNA with either the CMV or CAG promoters, used for immunizations. The observed luciferase activity in this comparison demonstrated that the CAG promoter-containing plasmid pCAGGS induced prolonged constitutive expression of MRP4 and an increased anti-MRP4 specific immune response even when the plasmid was injected less frequently. The method described here is one that can be broadly applicable as a general immunization strategy to develop antibodies against multi-transmembrane proteins, as well as target antigens that are difficult to express or purify in native and functionally active conformation.
Assuntos
Anticorpos/imunologia , Imunização , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Plasmídeos , Vacinas de DNA , Animais , Linhagem Celular , DNA Complementar/imunologia , DNA Complementar/farmacologia , Humanos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Associadas à Resistência a Múltiplos Medicamentos/biossíntese , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Plasmídeos/imunologia , Plasmídeos/farmacologia , Estrutura Secundária de Proteína , Vacinas de DNA/imunologia , Vacinas de DNA/farmacologiaRESUMO
Receptor-interacting protein kinase 4 (RIPK4) is required for epidermal differentiation and is mutated in Bartsocas-Papas syndrome. RIPK4 binds to protein kinase C, but its signaling mechanisms are largely unknown. Ectopic RIPK4, but not catalytically inactive or Bartsocas-Papas RIPK4 mutants, induced accumulation of cytosolic ß-catenin and a transcriptional program similar to that caused by Wnt3a. In Xenopus embryos, Ripk4 synergized with coexpressed Xwnt8, whereas Ripk4 morpholinos or catalytic inactive Ripk4 antagonized Wnt signaling. RIPK4 interacted constitutively with the adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 by RIPK4 favored canonical Wnt signaling. Wnt-dependent growth of xenografted human tumor cells was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Via de Sinalização Wnt , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , Proteínas Desgrenhadas , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Transplante de Neoplasias , Neoplasias/metabolismo , Neoplasias Ovarianas/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/genética , Transplante Heterólogo , Proteína Wnt3A/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/embriologia , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
Hepatitis C virus (HCV) infection is a major cause of liver disease and hepatocellular carcinoma. Glycan shielding has been proposed to be a mechanism by which HCV masks broadly neutralizing epitopes on its viral glycoproteins. However, the role of altered glycosylation in HCV resistance to broadly neutralizing antibodies is not fully understood. Here, we have generated potent HCV neutralizing antibodies hu5B3.v3 and MRCT10.v362 that, similar to the previously described AP33 and HCV1, bind to a highly conserved linear epitope on E2. We utilize a combination of in vitro resistance selections using the cell culture infectious HCV and structural analyses to identify mechanisms of HCV resistance to hu5B3.v3 and MRCT10.v362. Ultra deep sequencing from in vitro HCV resistance selection studies identified resistance mutations at asparagine N417 (N417S, N417T and N417G) as early as 5days post treatment. Comparison of the glycosylation status of soluble versions of the E2 glycoprotein containing the respective resistance mutations revealed a glycosylation shift from N417 to N415 in the N417S and N417T E2 proteins. The N417G E2 variant was glycosylated neither at residue 415 nor at residue 417 and remained sensitive to MRCT10.v362. Structural analyses of the E2 epitope bound to hu5B3.v3 Fab and MRCT10.v362 Fab using X-ray crystallography confirmed that residue N415 is buried within the antibody-peptide interface. Thus, in addition to previously described mutations at N415 that abrogate the ß-hairpin structure of this E2 linear epitope, we identify a second escape mechanism, termed glycan shifting, that decreases the efficacy of broadly neutralizing HCV antibodies.
Assuntos
Anticorpos Neutralizantes/imunologia , Hepacivirus/imunologia , Anticorpos Anti-Hepatite C/imunologia , Evasão da Resposta Imune , Polissacarídeos/imunologia , Processamento de Proteína Pós-Traducional , Proteínas do Envelope Viral/imunologia , Anticorpos Monoclonais/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Hepacivirus/química , Hepacivirus/genética , Sequenciamento de Nucleotídeos em Larga Escala , Polissacarídeos/metabolismo , Conformação Proteica , RNA Viral/genética , Proteínas do Envelope Viral/química , Proteínas do Envelope Viral/genéticaRESUMO
Canonical Wnt signaling is controlled intracellularly by the level of ß-catenin protein, which is dependent on Axin scaffolding of a complex that phosphorylates ß-catenin to target it for ubiquitylation and proteasomal degradation. This function of Axin is counteracted through relocalization of Axin protein to the Wnt receptor complex to allow for ligand-activated Wnt signaling. AXIN1 and AXIN2 protein levels are regulated by tankyrase-mediated poly(ADP-ribosyl)ation (PARsylation), which destabilizes Axin and promotes signaling. Mechanistically, how tankyrase limits Axin protein accumulation, and how tankyrase levels and activity are regulated for this function, are currently under investigation. By RNAi screening, we identified the RNF146 RING-type ubiquitin E3 ligase as a positive regulator of Wnt signaling that operates with tankyrase to maintain low steady-state levels of Axin proteins. RNF146 also destabilizes tankyrases TNKS1 and TNKS2 proteins and, in a reciprocal relationship, tankyrase activity reduces RNF146 protein levels. We show that RNF146, tankyrase, and Axin form a protein complex, and that RNF146 mediates ubiquitylation of all three proteins to target them for proteasomal degradation. RNF146 is a cytoplasmic protein that also prevents tankyrase protein aggregation at a centrosomal location. Tankyrase auto-PARsylation and PARsylation of Axin is known to lead to proteasome-mediated degradation of these proteins, and we demonstrate that, through ubiquitylation, RNF146 mediates this process to regulate Wnt signaling.
Assuntos
Proteína Axina/metabolismo , Transdução de Sinais , Tanquirases/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas Wnt/metabolismo , Centrossomo/metabolismo , Células HEK293 , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica , Transporte Proteico , Proteólise , UbiquitinaçãoRESUMO
Hedgehog (Hh) signaling in vertebrates depends on intraflagellar transport (IFT) within primary cilia. The Hh receptor Patched is found in cilia in the absence of Hh and is replaced by the signal transducer Smoothened within an hour of Hh stimulation. By generating antibodies capable of detecting endogenous pathway transcription factors Gli2 and Gli3, we monitored their kinetics of accumulation in cilia upon Hh stimulation. Localization occurs within minutes of Hh addition, making it the fastest reported readout of pathway activity, which permits more precise temporal and spatial localization of Hh signaling events. We show that the species of Gli3 that accumulates at cilium tips is full-length and likely not protein kinase A phosphorylated. We also confirmed that phosphorylation and betaTrCP/Cul1 are required for endogenous Gli3 processing and that this is inhibited by Hh. Surprisingly, however, Hh-dependent inhibition of processing does not lead to accumulation of full-length Gli3, but instead renders it labile, leading to its proteasomal degradation via the SPOP/Cul3 complex. In fact, full-length Gli3 disappears with faster kinetics than the Gli3 repressor, the latter not requiring SPOP/Cul3 or betaTrCP/Cul1. This may contribute to the increased Gli3 activator/repressor ratios found in IFT mutants.
Assuntos
Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Transdução de Sinais/fisiologia , Animais , Linhagem Celular , Cílios/ultraestrutura , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Hedgehog/genética , Humanos , Fatores de Transcrição Kruppel-Like/genética , Camundongos , Camundongos Endogâmicos BALB C , Proteínas do Tecido Nervoso/genética , Receptores Patched , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína Gli2 com Dedos de Zinco , Proteína Gli3 com Dedos de ZincoRESUMO
Antibodies directed against B cells are in use for the treatment of non-Hodgkin's lymphoma and autoimmune disorders. The B-cell-restricted surface antigen CD79b, a signaling component of the B-cell receptor, has been shown as a promising antibody target in mouse efficacy models of systemic lupus erythematosus. Anti-CD79b antibody-drug conjugates (ADC), cytotoxic drugs linked through specialized chemical linkers to antibodies, are effective in mouse xenograft models of non-Hodgkin's lymphoma. We were interested in evaluating the systemic effects of anti-CD79b antibodies and ADCs in normal animals as a step toward the development of these molecules as therapeutics. As we were unable to identify any cell surface binding anti-human CD79b antibodies that were cross-reactive to other species, we developed an antibody to cynomolgus monkey (Macaca fascicularis) CD79b (anti-cyCD79b). The anti-cynomolgus antibody, anti-cyCD79b (10D10), and the maytansine (tubulin inhibitor)-conjugated ADC, anti-cyCD79b (10D10)-MCC-DM1, were administered to cynomolgus monkeys at approximately 30 mg/kg (6,000 microg DM1/m(2)) for two doses 3 weeks apart. Anti-cyCD79b and anti-cyCD79b-MCC-DM1 resulted in peripheral blood B-cell depletion of approximately 65% and approximately 94%, respectively. In addition, anti-cyCD79b-MCC-DM1 resulted in near-complete absence of splenic germinal centers, an observation supporting an effect on dividing B cells. Both molecules were well tolerated, with minimal findings for the antibody and findings for the ADC limited to the lymphoid and hematopoietic systems, liver, and peripheral nerves. These preclinical data suggest that targeting CD79b with antibodies or ADCs may provide safe and effective therapies for B-cell malignancies and autoimmune diseases.
Assuntos
Anticorpos/imunologia , Antineoplásicos/farmacologia , Antígenos CD79/imunologia , Sequência de Aminoácidos , Animais , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Antígenos CD79/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Reações Cruzadas/efeitos dos fármacos , Citometria de Fluxo , Humanos , Tolerância Imunológica/efeitos dos fármacos , Macaca fascicularis/sangue , Macaca fascicularis/imunologia , Maitansina/farmacologia , Camundongos , Dados de Sequência Molecular , Baço/efeitos dos fármacos , Baço/imunologia , Baço/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Targeting cytotoxic drugs to cancer cells using antibody-drug conjugates (ADCs), particularly those with stable linkers between the drug and the antibody, could be an effective cancer treatment with low toxicity. However, for stable-linker ADCs to be effective, they must be internalized and degraded, limiting potential targets to surface antigens that are trafficked to lysosomes. CD79a and CD79b comprise the hetrodimeric signaling component of the B-cell receptor, and are attractive targets for the use of ADCs because they are B-cell-specific, expressed in non-Hodgkin lymphomas (NHL), and are trafficked to a lysosomal-like compartment as part of antigen presentation. We show here that the stable-linker ADCs anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are capable of target-dependent killing of nonHodgkin lymphoma cell lines in vitro. Further, these 2 ADCs are equally effective as low doses in xenograft models of follicular, mantle cell, and Burkitt lymphomas, even though several of these cell lines express relatively low levels of CD79b in vivo. In addition, we demonstrate that anti-CD79b ADCs were more effective than anti-CD79a ADCs and that, as hypothesized, anti-CD79b antibodies downregulated surface B-cell receptor and were trafficked to the lysosomal-like major histocompatibility complex class II-positive compartment MIIC. These results suggest that anti-CD79b-MCC-DM1 and anti-CD79b-MC-MMAF are promising therapeutics for the treatment of NHL.
Assuntos
Anticorpos/uso terapêutico , Antígenos CD79/imunologia , Imunoconjugados/uso terapêutico , Linfoma não Hodgkin/imunologia , Animais , Células CHO , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Feminino , Citometria de Fluxo , Antígenos HLA-D/imunologia , Humanos , Lisossomos/imunologia , Camundongos , Camundongos SCID , Receptores de Antígenos de Linfócitos B/imunologia , Transplante HeterólogoRESUMO
A new family of Ig domain receptors referred to as the immune receptor translocation-associated (IRTA) proteins, FcR homologs (FcRHs) or FcR-like that are expressed in lymphoid cells has been recently described. RNA expression analysis suggests that FcRH1-5/IRTA1-5 are expressed exclusively in subsets of the B-cell compartment. We generated mAbs to FcRH1-5/IRTA1-5 and examined their protein expression pattern in normal tissue and in chronic lymphocytic leukemia (CLL) cells. Our data indicated that FcRH1-5/IRTA1-5 were expressed in B-cell sub-populations; however, in some cases, the protein was not expressed in the same B-cell populations as suggested by the RNA expression analysis. FcRH1/IRTA5 was expressed throughout the B-cell lineage starting at the pro-B-cell stage but was down-regulated in plasma cells. FcRH2/IRTA4 was expressed preferentially in memory B cells. FcRH3/IRTA3 was expressed at low levels in naive, germinal center (GC) and memory B cells but was also expressed in NK cells. FcRH4/IRTA1 was expressed in a sub-population of memory B cells associated with mucosal tissue. FcRH5/IRTA2 was expressed in mature B cells and memory B cells and down-regulated in GC cells and, unlike all other B-cell-specific markers, maintained its expression in plasma cells from tonsil, spleen and bone marrow. We examined the expression of FcRH1-5/IRTA1-5 on the surface of CLL cells and found a similar pattern of expression on CLL cells as in the normal mature B cells, except for FcRH3/IRTA3 which was up-regulated in CLL.