ABSTRACT
Plasma membrane rupture (PMR) in dying cells undergoing pyroptosis or apoptosis requires the cell-surface protein NINJ11. PMR releases pro-inflammatory cytoplasmic molecules, collectively called damage-associated molecular patterns (DAMPs), that activate immune cells. Therefore, inhibiting NINJ1 and PMR may limit the inflammation that is associated with excessive cell death. Here we describe an anti-NINJ1 monoclonal antibody that specifically targets mouse NINJ1 and blocks oligomerization of NINJ1, preventing PMR. Electron microscopy studies showed that this antibody prevents NINJ1 from forming oligomeric filaments. In mice, inhibition of NINJ1 or Ninj1 deficiency ameliorated hepatocellular PMR induced with TNF plus D-galactosamine, concanavalin A, Jo2 anti-Fas agonist antibody or ischaemia-reperfusion injury. Accordingly, serum levels of lactate dehydrogenase, the liver enzymes alanine aminotransaminase and aspartate aminotransferase, and the DAMPs interleukin 18 and HMGB1 were reduced. Moreover, in the liver ischaemia-reperfusion injury model, there was an attendant reduction in neutrophil infiltration. These data indicate that NINJ1 mediates PMR and inflammation in diseases driven by aberrant hepatocellular death.
Subject(s)
Antibodies, Monoclonal , Cell Membrane , Inflammation , Liver , Nerve Growth Factors , Reperfusion Injury , Animals , Mice , Alanine Transaminase , Alarmins , Antibodies, Monoclonal/immunology , Aspartate Aminotransferases , Cell Adhesion Molecules, Neuronal/antagonists & inhibitors , Cell Adhesion Molecules, Neuronal/deficiency , Cell Adhesion Molecules, Neuronal/immunology , Cell Adhesion Molecules, Neuronal/ultrastructure , Cell Death , Cell Membrane/pathology , Cell Membrane/ultrastructure , Concanavalin A , Galactosamine , Hepatocytes/pathology , Hepatocytes/ultrastructure , Inflammation/pathology , Lactate Dehydrogenases , Liver/pathology , Microscopy, Electron , Nerve Growth Factors/antagonists & inhibitors , Nerve Growth Factors/deficiency , Nerve Growth Factors/immunology , Nerve Growth Factors/ultrastructure , Neutrophil Infiltration , Reperfusion Injury/pathologyABSTRACT
Recent single-cell studies of cancer in both mice and humans have identified the emergence of a myofibroblast population specifically marked by the highly restricted leucine-rich-repeat-containing protein 15 (LRRC15)1-3. However, the molecular signals that underlie the development of LRRC15+ cancer-associated fibroblasts (CAFs) and their direct impact on anti-tumour immunity are uncharacterized. Here in mouse models of pancreatic cancer, we provide in vivo genetic evidence that TGFß receptor type 2 signalling in healthy dermatopontin+ universal fibroblasts is essential for the development of cancer-associated LRRC15+ myofibroblasts. This axis also predominantly drives fibroblast lineage diversity in human cancers. Using newly developed Lrrc15-diphtheria toxin receptor knock-in mice to selectively deplete LRRC15+ CAFs, we show that depletion of this population markedly reduces the total tumour fibroblast content. Moreover, the CAF composition is recalibrated towards universal fibroblasts. This relieves direct suppression of tumour-infiltrating CD8+ T cells to enhance their effector function and augments tumour regression in response to anti-PDL1 immune checkpoint blockade. Collectively, these findings demonstrate that TGFß-dependent LRRC15+ CAFs dictate the tumour-fibroblast setpoint to promote tumour growth. These cells also directly suppress CD8+ T cell function and limit responsiveness to checkpoint blockade. Development of treatments that restore the homeostatic fibroblast setpoint by reducing the population of pro-disease LRRC15+ myofibroblasts may improve patient survival and response to immunotherapy.
Subject(s)
Cancer-Associated Fibroblasts , Membrane Proteins , Myofibroblasts , Pancreatic Neoplasms , Stromal Cells , Animals , Humans , Mice , Cancer-Associated Fibroblasts/metabolism , CD8-Positive T-Lymphocytes/immunology , Membrane Proteins/metabolism , Myofibroblasts/metabolism , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Receptors, Transforming Growth Factor beta , Transforming Growth Factor beta/metabolism , Tumor Microenvironment , B7-H1 AntigenABSTRACT
Inositol requiring enzyme 1 (IRE1) mitigates endoplasmic-reticulum (ER) stress by orchestrating the unfolded-protein response (UPR). IRE1 spans the ER membrane, and signals through a cytosolic kinase-endoribonuclease module. The endoribonuclease generates the transcription factor XBP1s by intron excision between similar RNA stem-loop endomotifs, and depletes select cellular mRNAs through regulated IRE1-dependent decay (RIDD). Paradoxically, in mammals RIDD seems to target only mRNAs with XBP1-like endomotifs, while in flies RIDD exhibits little sequence restriction. By comparing nascent and total IRE1α-controlled mRNAs in human cells, we identify not only canonical endomotif-containing RIDD substrates, but also targets without such motifs-degraded by a process we coin RIDDLE, for RIDD lacking endomotif. IRE1α displays two basic endoribonuclease modalities: highly specific, endomotif-directed cleavage, minimally requiring dimers; and more promiscuous, endomotif-independent processing, requiring phospho-oligomers. An oligomer-deficient IRE1α mutant fails to support RIDDLE in vitro and in cells. Our results advance current mechanistic understanding of the UPR.
Subject(s)
Endoplasmic Reticulum Stress , Endoplasmic Reticulum/metabolism , Endoribonucleases/metabolism , Protein Serine-Threonine Kinases/metabolism , Endoplasmic Reticulum/genetics , Endoribonucleases/genetics , Humans , Protein Serine-Threonine Kinases/genetics , RNA Stability , RNA, Messenger/genetics , RNA, Messenger/metabolism , Unfolded Protein ResponseABSTRACT
The cancer-associated fibroblast (CAF) marker podoplanin (PDPN) is generally correlated with poor clinical outcomes in cancer patients and thus represents a promising therapeutic target. Despite its biomedical relevance, basic aspects of PDPN biology such as its cellular functions and cell surface ligands remain poorly uncharacterized, thus challenging drug development. Here, we utilize a high throughput platform to elucidate the PDPN cell surface interactome, and uncover the neutrophil protein CD177 as a new binding partner. Quantitative proteomics analysis of the CAF phosphoproteome reveals a role for PDPN in cell signaling, growth and actomyosin contractility, among other processes. Moreover, cellular assays demonstrate that CD177 is a functional antagonist, recapitulating the phenotype observed in PDPN-deficient CAFs. In sum, starting from the unbiased elucidation of the PDPN co-receptome, our work provides insights into PDPN functions and reveals the PDPN/CD177 axis as a possible modulator of fibroblast physiology in the tumor microenvironment.
Subject(s)
Biomarkers, Tumor/metabolism , Cancer-Associated Fibroblasts/pathology , Colorectal Neoplasms/pathology , Gene Expression Regulation, Neoplastic , Isoantigens/metabolism , Membrane Glycoproteins/metabolism , Receptors, Cell Surface/metabolism , Tumor Microenvironment , Apoptosis , Biomarkers, Tumor/genetics , Cancer-Associated Fibroblasts/immunology , Cancer-Associated Fibroblasts/metabolism , Cell Proliferation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/immunology , Colorectal Neoplasms/metabolism , GPI-Linked Proteins/genetics , GPI-Linked Proteins/metabolism , Humans , Isoantigens/genetics , Membrane Glycoproteins/genetics , Neutrophils/immunology , Neutrophils/metabolism , Prognosis , Receptors, Cell Surface/genetics , Survival Rate , Tumor Cells, CulturedABSTRACT
Defective autophagy is strongly associated with chronic inflammation. Loss-of-function of the core autophagy gene Atg16l1 increases risk for Crohn's disease in part by enhancing innate immunity through myeloid cells such as macrophages. However, autophagy is also recognized as a mechanism for clearance of certain intracellular pathogens. These divergent observations prompted a re-evaluation of ATG16L1 in innate antimicrobial immunity. In this study, we found that loss of Atg16l1 in myeloid cells enhanced the killing of virulent Shigella flexneri (S.flexneri), a clinically relevant enteric bacterium that resides within the cytosol by escaping from membrane-bound compartments. Quantitative multiplexed proteomics of murine bone marrow-derived macrophages revealed that ATG16L1 deficiency significantly upregulated proteins involved in the glutathione-mediated antioxidant response to compensate for elevated oxidative stress, which simultaneously promoted S.flexneri killing. Consistent with this, myeloid-specific deletion of Atg16l1 in mice accelerated bacterial clearance in vitro and in vivo. Pharmacological induction of oxidative stress through suppression of cysteine import enhanced microbial clearance by macrophages. Conversely, antioxidant treatment of macrophages permitted S.flexneri proliferation. These findings demonstrate that control of oxidative stress by ATG16L1 and autophagy regulates antimicrobial immunity against intracellular pathogens.
Subject(s)
Autophagy-Related Proteins/deficiency , Autophagy , Dysentery, Bacillary/microbiology , Immunity, Innate , Macrophages/microbiology , Oxidative Stress , Proteome , Proteomics , Shigella flexneri/pathogenicity , Animals , Autophagy-Related Proteins/genetics , Cells, Cultured , Disease Models, Animal , Dysentery, Bacillary/immunology , Dysentery, Bacillary/metabolism , Host-Pathogen Interactions , Inflammation Mediators/metabolism , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred C57BL , Mice, Knockout , Microbial Viability , Shigella flexneri/immunology , Shigella flexneri/metabolism , VirulenceABSTRACT
Fibroblastic reticular cells (FRCs) are specialized stromal cells that define tissue architecture and regulate lymphocyte compartmentalization, homeostasis, and innate and adaptive immunity in secondary lymphoid organs (SLOs). In the present study, we used single-cell RNA sequencing (scRNA-seq) of human and mouse lymph nodes (LNs) to identify a subset of T cell-zone FRCs defined by the expression of Gremlin1 (Grem1) in both species. Grem1-CreERT2 knock-in mice enabled localization, multi-omics characterization and genetic depletion of Grem1+ FRCs. Grem1+ FRCs primarily localize at T-B cell junctions of SLOs, neighboring pre-dendritic cells and conventional dendritic cells (cDCs). As such, their depletion resulted in preferential loss and decreased homeostatic proliferation and survival of resident cDCs and compromised T cell immunity. Trajectory analysis of human LN scRNA-seq data revealed expression similarities to murine FRCs, with GREM1+ cells marking the endpoint of both trajectories. These findings illuminate a new Grem1+ fibroblastic niche in LNs that functions to maintain the homeostasis of lymphoid tissue-resident cDCs.
Subject(s)
Dendritic Cells, Follicular/immunology , Fibroblasts/immunology , Lymph Nodes/immunology , Stromal Cells/immunology , Aged , Animals , Apoptosis/genetics , Apoptosis/immunology , Cell Proliferation/genetics , Cell Survival/genetics , Cell Survival/immunology , Dendritic Cells, Follicular/metabolism , Female , Fibroblasts/metabolism , Gene Expression Regulation/immunology , Gene Knock-In Techniques , Humans , Immunity, Cellular/genetics , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Lymph Nodes/cytology , Male , Mice , Mice, Transgenic , RNA-Seq , Single-Cell Analysis , Stromal Cells/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolismABSTRACT
Antigen cross presentation, whereby exogenous antigens are presented by MHC class I molecules to CD8+ T cells, is essential for generating adaptive immunity to pathogens and tumor cells. Following endocytosis, it is widely understood that protein antigens must be transferred from endosomes to the cytosol where they are subject to ubiquitination and proteasome degradation prior to being translocated into the endoplasmic reticulum (ER), or possibly endosomes, via the TAP1/TAP2 complex. Revealing how antigens egress from endocytic organelles (endosome-to-cytosol transfer, ECT), however, has proved vexing. Here, we used two independent screens to identify the hydrogen peroxide-transporting channel aquaporin-3 (AQP3) as a regulator of ECT. AQP3 overexpression increased ECT, whereas AQP3 knockout or knockdown decreased ECT. Mechanistically, AQP3 appears to be important for hydrogen peroxide entry into the endosomal lumen where it affects lipid peroxidation and subsequent antigen release. AQP3-mediated regulation of ECT was functionally significant, as AQP3 modulation had a direct impact on the efficiency of antigen cross presentation in vitro. Finally, AQP3-/- mice exhibited a reduced ability to mount an anti-viral response and cross present exogenous extended peptide. Together, these results indicate that the AQP3-mediated transport of hydrogen peroxide can regulate endosomal lipid peroxidation and suggest that compromised membrane integrity and coordinated release of endosomal cargo is a likely mechanism for ECT.
Subject(s)
Aquaporin 3/metabolism , Cytosol/metabolism , Endosomes/metabolism , Animals , Antigen Presentation , Aquaporin 3/genetics , Biological Transport , Cells, Cultured , Gene Knockout Techniques , HEK293 Cells , Humans , Lipid Peroxidation , MiceABSTRACT
Estrogen receptor-positive (ER+) breast cancers frequently remain dependent on ER signaling even after acquiring resistance to endocrine agents, prompting the development of optimized ER antagonists. Fulvestrant is unique among approved ER therapeutics due to its capacity for full ER antagonism, thought to be achieved through ER degradation. The clinical potential of fulvestrant is limited by poor physicochemical features, spurring attempts to generate ER degraders with improved drug-like properties. We show that optimization of ER degradation does not guarantee full ER antagonism in breast cancer cells; ER "degraders" exhibit a spectrum of transcriptional activities and anti-proliferative potential. Mechanistically, we find that fulvestrant-like antagonists suppress ER transcriptional activity not by ER elimination, but by markedly slowing the intra-nuclear mobility of ER. Increased ER turnover occurs as a consequence of ER immobilization. These findings provide proof-of-concept that small molecule perturbation of transcription factor mobility may enable therapeutic targeting of this challenging target class.
Subject(s)
Breast Neoplasms/metabolism , Estrogen Receptor Antagonists/pharmacology , Fulvestrant/pharmacology , Receptors, Estrogen/antagonists & inhibitors , Receptors, Estrogen/metabolism , Animals , Breast Neoplasms/drug therapy , Breast Neoplasms/pathology , Cell Proliferation/drug effects , Cinnamates/pharmacology , Drug Resistance, Neoplasm , Estrogen Receptor Antagonists/therapeutic use , Female , Fulvestrant/therapeutic use , HEK293 Cells , Heterografts , Humans , Indazoles/pharmacology , Ligands , MCF-7 Cells , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Polymorphism, Single Nucleotide , Proteolysis/drug effects , Signal Transduction/drug effects , Transcription, Genetic/drug effectsABSTRACT
PURPOSE: The treatment of acute myeloid leukemia (AML) has not significantly changed in 40 years. Cytarabine- and anthracycline-based chemotherapy induction regimens (7 + 3) remain the standard of care, and most patients have poor long-term survival. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has demonstrated ADCs as a clinically validated option to enhance the effectiveness of induction therapy. We are interested in developing a next-generation ADC for AML to improve upon the initial success of Mylotarg. EXPERIMENTAL DESIGN: The expression pattern of CLL-1 and its hematopoietic potential were investigated. A novel anti-CLL-1-ADC, with a highly potent pyrrolobenzodiazepine (PBD) dimer conjugated through a self-immolative disulfide linker, was developed. The efficacy and safety profiles of this ADC were evaluated in mouse xenograft models and in cynomolgus monkeys. RESULTS: We demonstrate that CLL-1 shares similar prevalence and trafficking properties that make CD33 an excellent ADC target for AML, but lacks expression on hematopoietic stem cells that hampers current CD33-targeted ADCs. Our anti-CLL-1-ADC is highly effective at depleting tumor cells in AML xenograft models and lacks target independent toxicities at doses that depleted target monocytes and neutrophils in cynomolgus monkeys. CONCLUSIONS: Collectively, our data suggest that an anti-CLL-1-ADC has the potential to become an effective and safer treatment for AML in humans, by reducing and allowing for faster recovery from initial cytopenias than the current generation of ADCs for AML.
Subject(s)
Antibodies, Anti-Idiotypic/pharmacology , Immunoconjugates/pharmacology , Lectins, C-Type/immunology , Leukemia, Myeloid, Acute/drug therapy , Receptors, Mitogen/immunology , Animals , Flow Cytometry , Gene Expression Regulation, Neoplastic/drug effects , Humans , Lectins, C-Type/antagonists & inhibitors , Lectins, C-Type/genetics , Leukemia, Myeloid, Acute/genetics , Leukemia, Myeloid, Acute/immunology , Leukemia, Myeloid, Acute/pathology , Mice , Receptors, Mitogen/antagonists & inhibitors , Receptors, Mitogen/genetics , Sialic Acid Binding Ig-like Lectin 3/genetics , Sialic Acid Binding Ig-like Lectin 3/immunology , Xenograft Model Antitumor AssaysABSTRACT
There is a critical need for new antibacterial strategies to counter the growing problem of antibiotic resistance. In Gram-negative bacteria, the outer membrane (OM) provides a protective barrier against antibiotics and other environmental insults. The outer leaflet of the outer membrane is primarily composed of lipopolysaccharide (LPS). Outer membrane biogenesis presents many potentially compelling drug targets as this pathway is absent in higher eukaryotes. Most proteins involved in LPS biosynthesis and transport are essential; however, few compounds have been identified that inhibit these proteins. The inner membrane ABC transporter MsbA carries out the first essential step in the trafficking of LPS to the outer membrane. We conducted a biochemical screen for inhibitors of MsbA and identified a series of quinoline compounds that kill Escherichia coli through inhibition of its ATPase and transport activity, with no loss of activity against clinical multidrug-resistant strains. Identification of these selective inhibitors indicates that MsbA is a viable target for new antibiotics, and the compounds we identified serve as useful tools to further probe the LPS transport pathway in Gram-negative bacteria.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Bacterial Outer Membrane Proteins/metabolism , Bacterial Proteins/metabolism , Escherichia coli Proteins/metabolism , Escherichia coli/metabolism , Lipopolysaccharides/metabolism , Anti-Bacterial Agents/pharmacology , Biological Transport/drug effects , Biological Transport/physiology , Escherichia coli/drug effectsABSTRACT
Despite the recent success of antibody-drug conjugates (ADCs) in cancer therapy, a detailed understanding of their entry, trafficking, and metabolism in cancer cells is limited. To gain further insight into the activation mechanism of ADCs, we incorporated fluorescence resonance energy transfer (FRET) reporter groups into the linker connecting the antibody to the drug and studied various aspects of intracellular ADC processing mechanisms. When comparing the trafficking of the antibody-FRET drug conjugates in various different model cells, we found that the cellular background plays an important role in how the antigen-mediated antibody is processed. Certain tumor cells showed limited cytosolic transport of the payload despite efficient linker cleavage. Our FRET assay provides a facile and robust assessment of intracellular ADC activation that may have significant implications for the future development of ADCs.
Subject(s)
Biological Transport , Fluorescence Resonance Energy Transfer , Immunoconjugates/pharmacokinetics , Cell Membrane Permeability , Cross-Linking Reagents/chemistry , Humans , Immunoconjugates/metabolism , PeptidesABSTRACT
Therapeutic antibodies that block the programmed death-1 (PD-1)-programmed death-ligand 1 (PD-L1) pathway can induce robust and durable responses in patients with various cancers, including metastatic urothelial cancer. However, these responses only occur in a subset of patients. Elucidating the determinants of response and resistance is key to improving outcomes and developing new treatment strategies. Here we examined tumours from a large cohort of patients with metastatic urothelial cancer who were treated with an anti-PD-L1 agent (atezolizumab) and identified major determinants of clinical outcome. Response to treatment was associated with CD8+ T-effector cell phenotype and, to an even greater extent, high neoantigen or tumour mutation burden. Lack of response was associated with a signature of transforming growth factor ß (TGFß) signalling in fibroblasts. This occurred particularly in patients with tumours, which showed exclusion of CD8+ T cells from the tumour parenchyma that were instead found in the fibroblast- and collagen-rich peritumoural stroma; a common phenotype among patients with metastatic urothelial cancer. Using a mouse model that recapitulates this immune-excluded phenotype, we found that therapeutic co-administration of TGFß-blocking and anti-PD-L1 antibodies reduced TGFß signalling in stromal cells, facilitated T-cell penetration into the centre of tumours, and provoked vigorous anti-tumour immunity and tumour regression. Integration of these three independent biological features provides the best basis for understanding patient outcome in this setting and suggests that TGFß shapes the tumour microenvironment to restrain anti-tumour immunity by restricting T-cell infiltration.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , B7-H1 Antigen/antagonists & inhibitors , CD8-Positive T-Lymphocytes/drug effects , Transforming Growth Factor beta/metabolism , Urologic Neoplasms/drug therapy , Urologic Neoplasms/immunology , Urothelium/pathology , Animals , Antibodies/immunology , Antibodies/pharmacology , Antibodies/therapeutic use , Antibodies, Monoclonal, Humanized , Antigens, Neoplasm/analysis , Antigens, Neoplasm/immunology , Antigens, Neoplasm/metabolism , B7-H1 Antigen/immunology , CD8-Positive T-Lymphocytes/cytology , CD8-Positive T-Lymphocytes/immunology , Cell Cycle Checkpoints/drug effects , Cohort Studies , Collagen/metabolism , Disease Models, Animal , Drug Resistance, Neoplasm/drug effects , Fibroblasts/metabolism , Humans , Immunotherapy , Mice , Mutation , Neoplasm Metastasis , Phenotype , Signal Transduction/drug effects , Transforming Growth Factor beta/antagonists & inhibitors , Treatment Outcome , Tumor Microenvironment/immunology , Urologic Neoplasms/genetics , Urologic Neoplasms/pathology , Urothelium/drug effects , Urothelium/immunologyABSTRACT
Antibody-Drug Conjugates (ADCs) are a class of cancer therapeutics that combines antigen specificity and potent cytotoxicity in a single molecule as they are comprised of an engineered antibody linked chemically to a cytotoxic drug. Four ADCs have received approval by the Food and Drug Administration (FDA) and the European Medicine Agency (EMA) and can be prescribed for metastatic conditions while around 60 ADCs are currently enrolled in clinical trials. The efficacy of an ADC greatly relies on its intracellular trafficking and processing of its components to trigger tumor cell death. A limited number of studies have addressed these critical processes that both challenge and help foster the design of ADCs. This review highlights those mechanisms and their relevance for future development of ADCs as cancer therapeutics.
Subject(s)
Antineoplastic Agents, Immunological/metabolism , Antineoplastic Agents, Immunological/pharmacology , Immunoconjugates/metabolism , Immunoconjugates/pharmacology , Animals , Antineoplastic Agents, Immunological/chemistry , Antineoplastic Agents, Immunological/therapeutic use , Biological Transport , Biomarkers , Endocytosis , Humans , Immunoconjugates/chemistry , Immunoconjugates/therapeutic use , Metabolic Networks and Pathways , Molecular Targeted Therapy , Neoplasms/drug therapy , Neoplasms/genetics , Neoplasms/metabolism , Neoplasms/pathology , Tissue DistributionABSTRACT
Influenza A viruses (IAV) primarily target respiratory epithelial cells, but can also replicate in immune cells, including human dendritic cells (DCs). Super-resolution microscopy provides a novel method of visualizing viral trafficking by overcoming the resolution limit imposed by conventional light microscopy, without the laborious sample preparation of electron microscopy. Using three-color Stimulated Emission Depletion (STED) microscopy, we visualized input IAV nucleoprotein (NP), early and late endosomal compartments (EEA1 and LAMP1 respectively), and HLA-DR (DC membrane/cytosol) by immunofluorescence in human DCs. Surface bound IAV were internalized within 5 min of infection. The association of virus particles with early endosomes peaked at 5 min when 50% of NP+ signals were also EEA1+. Peak association with late endosomes occurred at 15 min when 60% of NP+ signals were LAMP1+. At 30 min of infection, the majority of NP signals were in the nucleus. Our findings illustrate that early IAV trafficking in human DCs proceeds via the classical endocytic pathway.
Subject(s)
Dendritic Cells/ultrastructure , Host-Pathogen Interactions , Influenza A virus/ultrastructure , Virion/ultrastructure , Dendritic Cells/metabolism , Dendritic Cells/virology , Endosomes/virology , Epithelial Cells/ultrastructure , Epithelial Cells/virology , HLA-DR Antigens/isolation & purification , Humans , Influenza A virus/genetics , Influenza A virus/isolation & purification , Influenza A virus/pathogenicity , Lysosomal Membrane Proteins/isolation & purification , Microscopy , Nucleocapsid Proteins , RNA-Binding Proteins/genetics , RNA-Binding Proteins/isolation & purification , Vesicular Transport Proteins/isolation & purification , Viral Core Proteins/genetics , Viral Core Proteins/isolation & purification , Virion/genetics , Virion/pathogenicity , Virus Replication/geneticsABSTRACT
Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.
Subject(s)
Cystine/metabolism , Peptides, Cyclic/metabolism , Protein Transport/physiology , Amino Acid Sequence , Animals , Cell Line, Tumor , Cell-Penetrating Peptides/metabolism , Cyclization/physiology , Cyclotides/metabolism , Endocytosis/physiology , Endosomes/metabolism , HeLa Cells , Humans , Lysosomes/metabolism , Mice , NIH 3T3 Cells , Plant Proteins/metabolismABSTRACT
Staphylococcus aureus is considered to be an extracellular pathogen. However, survival of S. aureus within host cells may provide a reservoir relatively protected from antibiotics, thus enabling long-term colonization of the host and explaining clinical failures and relapses after antibiotic therapy. Here we confirm that intracellular reservoirs of S. aureus in mice comprise a virulent subset of bacteria that can establish infection even in the presence of vancomycin, and we introduce a novel therapeutic that effectively kills intracellular S. aureus. This antibody-antibiotic conjugate consists of an anti-S. aureus antibody conjugated to a highly efficacious antibiotic that is activated only after it is released in the proteolytic environment of the phagolysosome. The antibody-antibiotic conjugate is superior to vancomycin for treatment of bacteraemia and provides direct evidence that intracellular S. aureus represents an important component of invasive infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Bacteremia , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Intracellular Space/microbiology , Staphylococcal Infections/microbiology , Staphylococcus aureus/drug effects , Vancomycin/pharmacology , Animals , Anti-Bacterial Agents/therapeutic use , Bacteremia/drug therapy , Bacteremia/microbiology , Carrier State/drug therapy , Carrier State/microbiology , Drug Design , Female , Immunoconjugates/chemistry , Intracellular Space/drug effects , Methicillin-Resistant Staphylococcus aureus/drug effects , Methicillin-Resistant Staphylococcus aureus/pathogenicity , Mice , Microbial Sensitivity Tests , Phagosomes/drug effects , Phagosomes/metabolism , Phagosomes/microbiology , Staphylococcal Infections/drug therapy , Staphylococcal Infections/pathology , Staphylococcus aureus/pathogenicity , Vancomycin/therapeutic useABSTRACT
B7-H4 has been implicated in cancers of the female reproductive system and investigated for its possible use as a biomarker for cancer, but there are no preclinical studies to demonstrate that B7-H4 is a molecular target for therapeutic intervention of cancer. We provide evidence that the prevalence and expression levels of B7-H4 are high in different subtypes of breast cancer and that only a few normal tissues express B7-H4 on the cell membrane. These profiles of low normal expression and upregulation in cancer provide an opportunity for the use of antibody-drug conjugates (ADCs), cytotoxic drugs chemically linked to antibodies, for the treatment of B7-H4 positive cancers. We have developed an ADC specific to B7-H4 that uses a linker drug consisting of a potent antimitotic, monomethyl auristatin E (MMAE), linked to engineered cysteines (THIOMAB) via a protease labile linker. We will refer to ADCs that use the THIOMAB format as TDCs to help distinguish the format from standard MC-vc-MMAE ADCs that are conjugated to the interchain disulfide bonds. Anti-B7-H4 (h1D11)-MC-vc-PAB-MMAE (h1D11 TDC) produced durable tumor regression in cell line and patient-derived xenograft models of triple-negative breast cancer. It also binds rat B7-H4 with similar affinity to human and allowed us to test for target dependent toxicity in rats. We found that our anti-B7-H4 TDC has toxicity findings similar to untargeted TDC. Our results validate B7-H4 as an ADC target for breast cancer and support the possible use of this TDC in the treatment of B7-H4(+) breast cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , Immunoconjugates/therapeutic use , Oligopeptides/therapeutic use , Animals , Antineoplastic Agents/chemistry , Blotting, Western , Cell Line, Tumor , Female , Flow Cytometry , Humans , Immunoconjugates/chemistry , Immunohistochemistry , Mice , Mice, SCID , Oligopeptides/chemistry , Rats , Rats, Sprague-Dawley , Triple Negative Breast Neoplasms/drug therapy , Xenograft Model Antitumor AssaysABSTRACT
Antibody-dependent cellular cytotoxicity (ADCC) is a key mechanism by which therapeutic antibodies mediate their antitumor effects. The absence of fucose on the heavy chain of the antibody increases the affinity between the antibody and FcγRIIIa, which results in increased in vitro and in vivo ADCC compared with the fucosylated form. However, the cellular and molecular mechanisms responsible for increased ADCC are unknown. Through a series of biochemical and cellular studies, we find that human natural killer (NK) cells stimulated with afucosylated antibody exhibit enhanced activation of proximal FcγRIIIa signaling and downstream pathways, as well as enhanced cytoskeletal rearrangement and degranulation, relative to stimulation with fucosylated antibody. Furthermore, analysis of the interaction between human NK cells and targets using a high-throughput microscope-based antibody-dependent cytotoxicity assay shows that afucosylated antibodies increase the number of NK cells capable of killing multiple targets and the rate with which targets are killed. We conclude that the increase in affinity between afucosylated antibodies and FcγRIIIa enhances activation of signaling molecules, promoting cytoskeletal rearrangement and degranulation, which, in turn, potentiates the cytotoxic characteristics of NK cells to increase efficiency of ADCC.