ABSTRACT
BACKGROUND: Dupilumab, a fully human monoclonal antibody that binds IL-4Rα and inhibits signaling of both IL-4 and IL-13, has shown efficacy across multiple diseases with underlying type 2 signatures and is approved for treatment of asthma, atopic dermatitis, and chronic sinusitis with nasal polyposis. We sought to provide a comprehensive analysis of the redundant and distinct roles of IL-4 and IL-13 in type 2 inflammation and report dupilumab mechanisms of action. METHODS: Using primary cell assays and a mouse model of house dust mite-induced asthma, we compared IL-4 vs IL-13 vs IL-4Rα blockers. RESULTS: Intranasal administration of either IL-4 or IL-13 confers an asthma-like phenotype in mice by inducing immune cell lung infiltration, including eosinophils, increasing cytokine/chemokine expression and mucus production, thus demonstrating redundant functions of these cytokines. We further teased out their respective contributions using human in vitro culture systems. Then, in a mouse asthma model by comparing in head-to-head studies, either IL-4 or IL-13 inhibition to dual IL-4/IL-13 inhibition, we demonstrate that blockade of both IL-4 and IL-13 is required to broadly block type 2 inflammation, which translates to protection from allergen-induced lung function impairment. Notably, only dual IL-4/IL-13 blockade prevented eosinophil infiltration into lung tissue without affecting circulating eosinophils, demonstrating that tissue, but not circulating eosinophils, contributes to disease pathology. CONCLUSIONS: Overall, these data support IL-4 and IL-13 as key drivers of type 2 inflammation and help provide insight into the therapeutic mechanism of dupilumab, a dual IL-4/IL-13 blocker, in multiple type 2 diseases.
Subject(s)
Interleukin-13 , Animals , Antibodies, Monoclonal, Humanized , Inflammation , Interleukin-4 , MiceABSTRACT
PURPOSE: Recent clinical data demonstrate that tumors harboring MET genetic alterations (exon 14 skip mutations and/or gene amplification) respond to small-molecule tyrosine kinase inhibitors, validating MET as a therapeutic target. Although antibody-mediated blockade of the MET pathway has not been successful in the clinic, the failures are likely the result of inadequate patient selection strategies as well as suboptimal antibody design. Thus, our goal was to generate a novel MET blocking antibody with enhanced efficacy. EXPERIMENTAL DESIGN: Here, we describe the activity of a biparatopic MET×MET antibody that recognizes two distinct epitopes in the MET Sema domain. We use a combination of in vitro assays and tumor models to characterize the effect of our antibody on MET signaling, MET intracellular trafficking, and the growth of MET-dependent cells/tumors. RESULTS: In MET-driven tumor models, our biparatopic antibody exhibits significantly better activity than either of the parental antibodies or the mixture of the two parental antibodies and outperforms several clinical-stage MET antibodies. Mechanistically, the biparatopic antibody inhibits MET recycling, thereby promoting lysosomal trafficking and degradation of MET. In contrast to the parental antibodies, the biparatopic antibody fails to activate MET-dependent biological responses, consistent with the observation that it recycles inefficiently and induces very transient downstream signaling. CONCLUSIONS: Our results provide strong support for the notion that biparatopic antibodies are a promising therapeutic modality, potentially having greater efficacy than that predicted from the properties of the parental antibodies.
Subject(s)
Antibodies, Monoclonal/pharmacology , Epitopes/immunology , Gene Amplification , Neoplasms/therapy , Proto-Oncogene Proteins c-met/metabolism , Animals , Cell Line, Tumor , Epitopes/genetics , Humans , Mice , Mice, SCID , Neoplasms/genetics , Neoplasms/immunology , Neoplasms/pathology , Protein Transport , Proto-Oncogene Proteins c-met/antagonists & inhibitors , Proto-Oncogene Proteins c-met/genetics , Xenograft Model Antitumor AssaysABSTRACT
Harnessing complement-mediated cytotoxicity by therapeutic antibodies has been limited because of dependency on size and density of antigen, structural constraints resulting from orientation of antibody binding, and blockade of complement activation by inhibitors expressed on target cells. We developed a modular bispecific antibody platform that directs the complement-initiating protein C1q to target cells, increases local complement deposition and induces cytotoxicity against target antigens with a wide-range of expression. The broad utility of this approach to eliminate both prokaryotic and eukaryotic cells was demonstrated by pairing a unique C1q-recruiting arm with multiple targeting arms specific for Staphylococcus aureus, Pseudomonas aeruginosa, B-cells and T-cells, indicating applicability for diverse indications ranging from infectious diseases to cancer. Generation of C1q humanized mice allowed for demonstration of the efficacy of this approach to clear disease-inducing cells in vivo. In summary, we present a novel, broadly applicable, and versatile therapeutic modality for targeted cell depletion.
Subject(s)
Antibodies, Bispecific/immunology , Complement System Proteins/immunology , Cytotoxicity, Immunologic , Animals , Antibody-Dependent Cell Cytotoxicity/immunology , Complement Activation , Complement Membrane Attack Complex/metabolism , Disease Models, Animal , Humans , Mice , Mice, Transgenic , Protein Binding , Staphylococcal Infections/immunology , Staphylococcal Infections/metabolism , Staphylococcal Infections/microbiology , Staphylococcus aureus/immunologyABSTRACT
Previously, it was shown that cationic alpha-peptides derived from the human immunodeficiency virus TAT protein transduction domain blocked herpes simplex virus type 1 (HSV-1) entry. We now show that cationic oligomers of beta-amino acids ("beta-peptides") inhibit HSV-1 infection. Among three cationic beta-peptides tested, the most effective inhibition was observed for the one with a strong propensity to adopt a helical conformation in which cationic and hydrophobic residues are segregated from one another ("globally amphiphilic helix"). The antiviral effect was not cell type specific. Inhibition of virus infection by the beta-peptides occurred at the postattachment penetration step, with a 50% effective concentration of 3 muM for the most-effective beta-peptide. The beta-peptides did not inactivate virions in solution, nor did they induce resistance to infection when cells were pretreated with the beta-peptides. The beta-peptides showed little if any toxicity toward Vero cells. These results raise the possibility that cationic beta-peptides may be useful antiviral agents for HSV-1 and demonstrate the potential of beta-peptides as novel antiviral drugs.
Subject(s)
Antiviral Agents/pharmacology , Herpesvirus 1, Human/drug effects , Peptides/pharmacology , Animals , Antiviral Agents/chemical synthesis , Antiviral Agents/chemistry , Cations/chemistry , Cells, Cultured , Chlorocebus aethiops , Cornea/cytology , Cornea/virology , Fibroblasts/virology , Herpesvirus 1, Human/pathogenicity , Humans , Microbial Sensitivity Tests/methods , Peptides/chemical synthesis , Peptides/chemistry , Protein Conformation , Vero CellsABSTRACT
Oxidative stress is a deleterious force that must be combated relentlessly by aerobic organisms and is known to underlie many human diseases including atherosclerosis, Parkinson's disease, and Alzheimer's disease. Information available about the oxidative stress response has come primarily from studies using reactive oxygen species (ROS) with ill-defined locations within the cell. Thus, existing models do not account for possible differences between stress originating within particular regions of the cell. Here, oxidative stress is studied at the subcellular level using ROS-generating compounds localizing within two different organelles: the nucleus and the mitochondrion. Differences in cytotoxicity, gene expression, and survival pathway activation are detected as a function of the subcellular origin of oxidative stress, indicating that independent mechanisms are used to cope with oxidative stress arising in different cellular compartments. These comparative studies, enabled by the development of organelle-specific oxidants, examine the cellular responses to site-specific oxidative stress with heightened precision.
Subject(s)
Organelles , Oxidative Stress , Peptides/metabolism , Cell Line , Flow Cytometry , Humans , Polymerase Chain Reaction , Reactive Oxygen Species/metabolismABSTRACT
The Prolactin Receptor (PRLR) is a type 1 cytokine receptor that is expressed in a subset of breast cancers and may contribute to its pathogenesis. It is relatively overexpressed in approximately 25% of human breast tumors while expressed at low levels in some normal human tissues including the mammary gland. We developed an anti-PRLR antibody-drug conjugate (ADC), to target PRLR-positive breast cancer. REGN2878-DM1 is comprised of a fully human high-affinity function-blocking anti-PRLR IgG1 antibody (REGN2878) conjugated via a noncleavable SMCC linker to the cytotoxic maytansine derivative DM1. Both unconjugated REGN2878 and conjugated REGN2878-DM1 block PRL-mediated activation in vitro and are rapidly internalized into lysosomes. REGN2878-DM1 induces potent cell-cycle arrest and cytotoxicity in PRLR-expressing tumor cell lines. In vivo, REGN2878-DM1 demonstrated significant antigen-specific antitumor activity against breast cancer xenograft models. In addition, REGN2878-DM1 showed additive activity when combined with the antiestrogen agent fulvestrant. These results illustrate promising antitumor activity against PRLR-positive breast cancer xenografts and support the evaluation of anti-PRLR ADCs as potential therapeutic agents in breast cancer. Mol Cancer Ther; 16(7); 1299-311. ©2017 AACR.
Subject(s)
Antibodies, Monoclonal, Humanized/administration & dosage , Breast Neoplasms/drug therapy , Immunoconjugates/administration & dosage , Receptors, Prolactin/immunology , Animals , Antibodies, Anti-Idiotypic/administration & dosage , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal, Humanized/immunology , Breast Neoplasms/genetics , Breast Neoplasms/immunology , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Female , Humans , Immunoconjugates/immunology , Mice , Receptors, Prolactin/genetics , Xenograft Model Antitumor AssaysABSTRACT
Bacteriorhodopsin (bR) is solubilized efficiently as a monomer by a novel surfactant, a tripod amphiphile (TPA), which permits the formation of purple hexagonal bR crystals under several conditions. The crystals, although small, diffract to 2.5 A resolution using synchrotron radiation. TPA may be useful for the solubilization, purification, and crystallization of other membrane proteins.
Subject(s)
Bacteriorhodopsins/chemistry , Halobacterium salinarum/chemistry , Surface-Active Agents/chemistry , Bacteriorhodopsins/isolation & purification , Crystallization , Crystallography, X-Ray , Purple Membrane/chemistryABSTRACT
BACKGROUND: Loss of skeletal muscle mass and function in humans is associated with significant morbidity and mortality. The role of myostatin as a key negative regulator of skeletal muscle mass and function has supported the concept that inactivation of myostatin could be a useful approach for treating muscle wasting diseases. METHODS: We generated a myostatin monoclonal blocking antibody (REGN1033) and characterized its effects in vitro using surface plasmon resonance biacore and cell-based Smad2/3 signaling assays. REGN1033 was tested in mice for the ability to induce skeletal muscle hypertrophy and prevent atrophy induced by immobilization, hindlimb suspension, or dexamethasone. The effect of REGN1033 on exercise training was tested in aged mice. Messenger RNA sequencing, immunohistochemistry, and ex vivo force measurements were performed on skeletal muscle samples from REGN1033-treated mice. RESULTS: The human monoclonal antibody REGN1033 is a specific and potent myostatin antagonist. Chronic treatment of mice with REGN1033 increased muscle fiber size, muscle mass, and force production. REGN1033 prevented the loss of muscle mass induced by immobilization, glucocorticoid treatment, or hindlimb unweighting and increased the gain of muscle mass during recovery from pre-existing atrophy. In aged mice, REGN1033 increased muscle mass and strength and improved physical performance during treadmill exercise. CONCLUSIONS: We show that specific myostatin antagonism with the human antibody REGN1033 enhanced muscle mass and function in young and aged mice and had beneficial effects in models of skeletal muscle atrophy.
ABSTRACT
EGFR blocking antibodies are approved for the treatment of colorectal cancer and head and neck squamous cell carcinoma (HNSCC). Although ERBB3 signaling has been proposed to limit the effectiveness of EGFR inhibitors, the underlying molecular mechanisms are not fully understood. To gain insight into these mechanisms, we generated potent blocking antibodies against ERBB3 (REGN1400) and EGFR (REGN955). We show that EGFR and ERBB3 are coactivated in multiple HNSCC cell lines and that combined blockade of EGFR and ERBB3 inhibits growth of these cell lines more effectively than blockade of either receptor alone. Blockade of EGFR with REGN955 strongly inhibited activation of ERK in HNSCC cell lines, whereas blockade of ERBB3 with REGN1400 strongly inhibited activation of Akt; only the combination of the 2 antibodies blocked both of these essential downstream pathways. We used a HER2 blocking antibody to show that ERBB3 phosphorylation in HNSCC and colorectal cancer cells is strictly dependent on association with HER2, but not EGFR, and that neuregulin 1 activates ERBB3/HER2 signaling to reverse the effect of EGFR blockade on colorectal cancer cell growth. Finally, although REGN1400 and REGN955 as single agents slowed the growth of HNSCC and colorectal cancer xenografts, the combination of REGN1400 plus REGN955 caused significant tumor regression. Our results indicate that activation of the Akt survival pathway by ERBB3/HER2 limits the effectiveness of EGFR inhibition, suggesting that REGN1400, which is currently in a phase I clinical trial, could provide benefit when combined with EGFR blocking antibodies.
Subject(s)
Colorectal Neoplasms/metabolism , Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , Head and Neck Neoplasms/metabolism , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Animals , Antibodies, Blocking/pharmacology , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized/pharmacology , Cell Line, Tumor , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Disease Models, Animal , Head and Neck Neoplasms/drug therapy , Head and Neck Neoplasms/pathology , Humans , Mice , Protein Binding , Proto-Oncogene Proteins c-akt/metabolism , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/antagonists & inhibitors , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
Short cationic oligomers, including arginine-rich peptides and analogous beta-amino acid oligomers ("beta-peptides"), can enter the cytoplasm and nucleus of a living cell from the extracellular medium. It seems increasingly clear that multiple entry pathways are possible, depending upon the structure of the guanidinium-rich molecule, the type of cell, and other factors. We have previously shown that conformational stability and spatial clustering of guanidinium groups increase the HeLa cell entry efficiency of short helical beta-peptides bearing six guanidinium groups, results that suggest that these beta-peptides could be useful tools for studying the entry process. Here we describe studies intended to identify the point in the entry process at which helix stability and spatial arrangement of guanidinium groups exert their effect. Our results suggest that key distinctions involve the mode of interaction between different guanidinium-rich beta-peptides and the HeLa cell surface. A specific guanidinium display appears to be required for proper engagement of cell-surface heparan sulfate proteoglycans and concomitant induction of endocytic uptake.
Subject(s)
Guanidine/chemistry , Peptides/chemistry , Cations/chemistry , Cell Adhesion/physiology , Endocytosis/drug effects , Epidermal Growth Factor/chemistry , Epidermal Growth Factor/pharmacology , HeLa Cells , Humans , Lipid Bilayers/chemistry , Liposomes/chemistry , Molecular Conformation , Peptides/pharmacology , Time FactorsABSTRACT
We have used our ability to control beta-peptide secondary structure in order to explore the effects of conformational stability and geometry of guanidinium display on cell entry. Both of these factors affect the rate and relative amount of beta-peptide accumulation in the cytoplasm and nucleus of live HeLa cells. These beta-peptides do not show significant differences in cell surface binding, implying that structure and guanidinium display are important in a later step in cell entry than initial surface binding.
Subject(s)
Arginine/chemistry , Arginine/metabolism , Guanidine/chemistry , Guanidine/metabolism , Oligopeptides/chemistry , Oligopeptides/metabolism , Endocytosis , HeLa Cells , Humans , Protein Conformation , Protein Structure, Secondary , Structure-Activity RelationshipABSTRACT
Several short, highly cationic peptides are able to enter the cytoplasm and nucleus of cells from the extracellular medium. The mechanism of entry is unknown. A number of fluorescence-based studies suggested that these molecules cross the plasma membrane by an energy-independent process, directly gaining access to the cytoplasm. Recent reports have questioned this conclusion, attributing the prior observations to artifacts resulting from fixation procedures used to prepare cells for fluorescence microscopy. These studies analyzed live cells and showed that the peptides entered through endocytosis and accumulated in endocytic vesicles, without necessarily entering the cytoplasm. To resolve this controversy and to extend the analyses to non-natural beta-peptide sequences, we studied the cytoplasmic and nuclear delivery of a fluorescein-labeled 9-residue sequence derived from the human immunodeficiency virus transactivator of transcription (TAT) peptide, TAT-(47-57), as well as a similarly labeled 12-residue beta-peptide, beta-(VRR)4, in live cells. Using fluorescence confocal microscopy, we show that when added to cells, both peptides are found in endocytic vesicles containing the transferrin receptor as well as in the cytoplasm and nucleus (TAT-(47-57)) or nucleolus (beta-(VRR)4). The cells were verified to be intact through all experimental procedures by demonstrating their ability to exclude propidium iodide. Endocytic entry of the peptides was blocked by the energy poisons sodium azide and 2-deoxyglucose, whereas staining of the nucleus (nucleolus), but not endocytic vesicles, was abrogated by treating the cells with ammonium chloride. Our observations are consistent with the proposal that TAT-(47-57) and beta-(VRR)4 enter cells by endocytosis and then exit an endosomal compartment to enter the cytoplasm by means of a mechanism requiring endosome acidification.