ABSTRACT
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.
Subject(s)
Neoplasms/blood supply , Neovascularization, Pathologic , Pregnancy Proteins/metabolism , Animals , Antibodies, Monoclonal/pharmacology , Cell Line, Tumor , Humans , Mice , Mice, Inbred BALB C , Placenta Growth Factor , Pregnancy Proteins/antagonists & inhibitors , Vascular Endothelial Growth FactorsABSTRACT
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.
Subject(s)
Cell Differentiation/drug effects , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Molecular Targeted Therapy , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/pathology , Receptor-Like Protein Tyrosine Phosphatases, Class 2/metabolism , Thrombospondins/metabolism , Animals , Antibodies/immunology , Antibodies/pharmacology , Antibodies/therapeutic use , Cell Division/drug effects , Colorectal Neoplasms/metabolism , Disease Progression , Female , Gene Expression Regulation/drug effects , Humans , Intestinal Mucosa/metabolism , Intestines/cytology , Intestines/drug effects , Intestines/pathology , Male , Mice , Neoplastic Stem Cells/metabolism , Stem Cells/cytology , Stem Cells/metabolism , Thrombospondins/antagonists & inhibitors , Thrombospondins/immunology , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Tumor Suppressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Amino Acid Motifs , Animals , Carrier Proteins/metabolism , Cell Line , Cell Line, Tumor , Cell Proliferation , Cell Transformation, Neoplastic , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Male , Mice , Nuclear Proteins/deficiency , PTEN Phosphohydrolase/deficiency , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/deficiency , Ubiquitin-Protein Ligases/genetics , UbiquitinationABSTRACT
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.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/pharmacology , Antibodies, Neutralizing/pharmacology , Antibodies, Viral/pharmacology , Cytomegalovirus Infections/congenital , Cytomegalovirus Infections/drug therapy , Cytomegalovirus/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Cytomegalovirus Infections/immunology , Cytomegalovirus Infections/pathology , Disease Models, Animal , Guinea Pigs , HEK293 Cells , HumansABSTRACT
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.
Subject(s)
Homeostasis/immunology , Macrophages, Peritoneal/immunology , Membrane Proteins/metabolism , Receptors, Cell Surface/metabolism , Animals , Antibodies/immunology , Antibody Formation/immunology , Apoptosis/immunology , Cell Adhesion , Cell Count , Cell Line , Erythrocytes/immunology , Humans , Hypersensitivity, Delayed/immunology , Macrophages, Peritoneal/cytology , Membrane Proteins/deficiency , Mice , Phagocytosis/immunology , Protein Transport , Receptors, Complement/immunology , Sheep , Spleen/cytology , Spleen/immunology , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
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.
Subject(s)
Membrane Proteins/antagonists & inhibitors , Membrane Proteins/metabolism , Neoplasms/blood supply , Neoplasms/pathology , Neovascularization, Pathologic , Signal Transduction , Animals , Cell Differentiation , Cell Line , Cell Proliferation , Endothelium, Vascular/cytology , Homeostasis , Humans , Intestine, Small/cytology , Intestine, Small/metabolism , Intracellular Signaling Peptides and Proteins , Mice , Receptors, Notch/metabolism , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Vascular Endothelial Growth Factor A/metabolismABSTRACT
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.
Subject(s)
Breast Neoplasms/drug therapy , Glial Cell Line-Derived Neurotrophic Factor Receptors/antagonists & inhibitors , Immunoconjugates/pharmacology , Xenograft Model Antitumor Assays , Animals , Antibodies/chemistry , Antibodies/immunology , Antibodies/pharmacology , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/genetics , Female , Gene Expression Regulation, Neoplastic/drug effects , Glial Cell Line-Derived Neurotrophic Factor Receptors/genetics , Glial Cell Line-Derived Neurotrophic Factor Receptors/immunology , HEK293 Cells , Humans , Immunoconjugates/immunology , Immunoconjugates/pharmacokinetics , MCF-7 Cells , Macaca fascicularis , Mice, Nude , Mice, SCID , Rats, Sprague-Dawley , Receptors, Steroid/metabolism , Tumor Burden/drug effects , Tumor Burden/geneticsABSTRACT
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.
Subject(s)
Antibodies, Bispecific/biosynthesis , Antibodies, Monoclonal/biosynthesis , High-Throughput Screening Assays , Immunoglobulin G/biosynthesis , Protein Engineering/methods , Animals , Antibodies, Bispecific/chemistry , Antibodies, Bispecific/genetics , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , CHO Cells , Cloning, Molecular , Cricetulus , Fibroblast Growth Factors/genetics , Fibroblast Growth Factors/immunology , Gene Expression , Genetic Vectors/chemistry , Genetic Vectors/metabolism , HEK293 Cells , Humans , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Klotho Proteins , Membrane Proteins/genetics , Membrane Proteins/immunology , Mice , Mice, Inbred BALB C , Receptor, Fibroblast Growth Factor, Type 1/genetics , Receptor, Fibroblast Growth Factor, Type 1/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
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.
Subject(s)
Antibody-Dependent Cell Cytotoxicity , Asthma/therapy , Th2 Cells/cytology , Animals , Antibodies, Monoclonal, Humanized/immunology , Basophils/cytology , Cytokines , Disease Models, Animal , Eosinophils/cytology , Humans , Immunity, Innate , Lung/cytology , Lung/immunology , Lymphocytes/cytology , Lymphoid Tissue/cytology , Lymphoid Tissue/immunology , Mice , Mice, SCID , Mice, TransgenicABSTRACT
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.
Subject(s)
Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Colonic Neoplasms/drug therapy , Immunotoxins/pharmacology , Neoplastic Stem Cells/drug effects , Receptors, G-Protein-Coupled/immunology , Animals , Antineoplastic Agents/immunology , Cell Line, Tumor , Cell Proliferation/drug effects , Colonic Neoplasms/genetics , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Dose-Response Relationship, Drug , Feasibility Studies , Female , Gene Expression Regulation, Neoplastic , Genes, APC , Immunotoxins/immunology , Immunotoxins/metabolism , Inhibitory Concentration 50 , Male , Mice, Inbred C57BL , Mice, Nude , Mice, Transgenic , Neoplastic Stem Cells/immunology , Neoplastic Stem Cells/metabolism , Neoplastic Stem Cells/pathology , Rats, Sprague-Dawley , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Time Factors , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antibodies/immunology , Immunization , Multidrug Resistance-Associated Proteins/immunology , Plasmids , Vaccines, DNA , Animals , Cell Line , DNA, Complementary/immunology , DNA, Complementary/pharmacology , Humans , Mice, Inbred BALB C , Mice, Knockout , Multidrug Resistance-Associated Proteins/biosynthesis , Multidrug Resistance-Associated Proteins/genetics , Plasmids/immunology , Plasmids/pharmacology , Protein Structure, Secondary , Vaccines, DNA/immunology , Vaccines, DNA/pharmacologyABSTRACT
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.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Phosphoproteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Wnt Signaling Pathway , Xenopus Proteins/metabolism , Animals , Cell Line , Cell Line, Tumor , Cytosol/metabolism , Dishevelled Proteins , Female , Gene Knockdown Techniques , HEK293 Cells , Humans , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Neoplasm Transplantation , Neoplasms/metabolism , Ovarian Neoplasms/metabolism , Phosphorylation , Protein Serine-Threonine Kinases/genetics , Transplantation, Heterologous , Wnt3A Protein/metabolism , Xenopus Proteins/genetics , Xenopus laevis/embryology , Xenopus laevis/metabolism , beta Catenin/metabolismABSTRACT
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.
Subject(s)
Antibodies, Neutralizing/immunology , Hepacivirus/immunology , Hepatitis C Antibodies/immunology , Immune Evasion , Polysaccharides/immunology , Protein Processing, Post-Translational , Viral Envelope Proteins/immunology , Antibodies, Monoclonal/immunology , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/immunology , Hepacivirus/chemistry , Hepacivirus/genetics , High-Throughput Nucleotide Sequencing , Polysaccharides/metabolism , Protein Conformation , RNA, Viral/genetics , Viral Envelope Proteins/chemistry , Viral Envelope Proteins/geneticsABSTRACT
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.
Subject(s)
Axin Protein/metabolism , Signal Transduction , Tankyrases/metabolism , Ubiquitin-Protein Ligases/metabolism , Wnt Proteins/metabolism , Centrosome/metabolism , HEK293 Cells , Humans , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Protein Transport , Proteolysis , UbiquitinationABSTRACT
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.
Subject(s)
Cilia/metabolism , Hedgehog Proteins/metabolism , Kruppel-Like Transcription Factors/metabolism , Nerve Tissue Proteins/metabolism , Signal Transduction/physiology , Animals , Cell Line , Cilia/ultrastructure , Cyclic AMP-Dependent Protein Kinases/metabolism , Hedgehog Proteins/genetics , Humans , Kruppel-Like Transcription Factors/genetics , Mice , Mice, Inbred BALB C , Nerve Tissue Proteins/genetics , Patched Receptors , Proteasome Endopeptidase Complex/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Smoothened Receptor , Zinc Finger Protein Gli2 , Zinc Finger Protein Gli3ABSTRACT
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.
Subject(s)
Antibodies/immunology , Antineoplastic Agents/pharmacology , CD79 Antigens/immunology , Amino Acid Sequence , Animals , Antibody Formation/drug effects , B-Lymphocytes/drug effects , B-Lymphocytes/immunology , CD79 Antigens/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cross Reactions/drug effects , Flow Cytometry , Humans , Immune Tolerance/drug effects , Macaca fascicularis/blood , Macaca fascicularis/immunology , Maytansine/pharmacology , Mice , Molecular Sequence Data , Spleen/drug effects , Spleen/immunology , Spleen/pathology , Xenograft Model Antitumor AssaysABSTRACT
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.
Subject(s)
Antibodies/therapeutic use , CD79 Antigens/immunology , Immunoconjugates/therapeutic use , Lymphoma, Non-Hodgkin/immunology , Animals , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Female , Flow Cytometry , HLA-D Antigens/immunology , Humans , Lysosomes/immunology , Mice , Mice, SCID , Receptors, Antigen, B-Cell/immunology , Transplantation, HeterologousABSTRACT
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.
Subject(s)
B-Lymphocytes/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Receptors, Cell Surface/biosynthesis , Receptors, Immunologic/biosynthesis , Animals , Flow Cytometry , Humans , Immunohistochemistry , Killer Cells, Natural/metabolism , Mice , Polymerase Chain Reaction , Receptors, FcABSTRACT
Vascular endothelial growth factor-A (VEGF) is an important regulator of vascular permeability. In preclinical studies, VEGF induces endothelial fenestrations in pre-existing and neo-vasculature, while inhibition of VEGF leads to a reduction in endothelial fenestrations. Recently, vascular regression in response to VEGF inhibition has been shown to correlate with the presence of endothelial fenestrations. Plasmalemmal vesicle-associated protein (PLVAP) is believed to be a component of diaphragmed endothelial fenestrations, but a direct relationship with VEGF signalling has not been established. The aim of this study was to characterize the expression pattern of PLVAP and investigate whether PLVAP is a transcriptional target of VEGF signal transduction. The expression pattern of PLVAP was characterized in normal and neoplastic human tissues by in situ hybridization and/or immunohistochemistry. The role of VEGF signal transduction in the regulation of PLVAP expression was investigated in vitro using receptor-selective engineered forms of VEGF, a neutralizing monoclonal antibody against VEGF, and inhibitors of downstream signalling pathways. PLVAP mRNA and protein were widely expressed in the endothelium of normal and neoplastic tissues. In cultured endothelial cells, VEGF signalling through receptor 2 stimulated expression of PLVAP total RNA and protein. This induction could be blocked with an anti-VEGF monoclonal antibody and by inhibitors of phosphatidylinositol 3-kinase (LY294002) or p38 mitogen-activated protein kinase (SB203580), but not by PD98059, a mitogen-activated protein/extracellular signal-regulated kinase 1 inhibitor. These data show that PLVAP is more widely expressed in the vasculature of normal tissues than previously thought and that it is expressed in the vasculature of most human tumours. We suggest that PLVAP is a downstream target of VEGF signalling. This work solidifies the association between VEGF and the appearance and maintenance of fenestrations by providing a potential mechanistic link.