ABSTRACT
Measuring responses in the proteome to various perturbations improves our understanding of biological systems. The value of information gained from such studies is directly proportional to the number of proteins measured. To overcome technical challenges associated with highly multiplexed measurements, we developed an affinity reagent-based method that uses aptamers with protein-like side chains along with an assay that takes advantage of their unique properties. As hybrid affinity reagents, modified aptamers are fully comparable to antibodies in terms of binding characteristics toward proteins, including epitope size, shape complementarity, affinity and specificity. Our assay combines these intrinsic binding properties with serial kinetic proofreading steps to allow highly effective partitioning of stable specific complexes from unstable nonspecific complexes. The use of these orthogonal methods to enhance specificity effectively overcomes the severe limitation to multiplexing inherent to the use of sandwich-based methods. Our assay currently measures half of the unique proteins encoded in the human genome with femtomolar sensitivity, broad dynamic range and exceptionally high reproducibility. Using machine learning to identify patterns of change, we have developed tests based on measurement of multiple proteins predictive of current health states and future disease risk to guide a holistic approach to precision medicine.
ABSTRACT
Early detection of cancer is vital for the best chance of successful treatment, but half of all cancers are diagnosed at an advanced stage. A simple and reliable blood screening test applied routinely would therefore address a major unmet medical need. To gain insight into the value of protein biomarkers in early detection and stratification of cancer we determined the time course of changes in the plasma proteome of mice carrying transplanted human lung, breast, colon, or ovarian tumors. For protein measurements we used an aptamer-based assay which simultaneously measures ~ 5000 proteins. Along with tumor lineage-specific biomarkers, we also found 15 markers shared among all cancer types that included the energy metabolism enzymes glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phophate isomerase and dihydrolipoyl dehydrogenase as well as several important biomarkers for maintaining protein, lipid, nucleotide, or carbohydrate balance such as tryptophanyl t-RNA synthetase and nucleoside diphosphate kinase. Using significantly altered proteins in the tumor bearing mice, we developed models to stratify tumor types and to estimate the minimum detectable tumor volume. Finally, we identified significantly enriched common and unique biological pathways among the eight tumor cell lines tested.
Subject(s)
Ovarian Neoplasms , Proteome , Female , Humans , Mice , Animals , Proteome/metabolism , Biomarkers, Tumor/metabolism , Energy Metabolism , Cell Line, TumorABSTRACT
Since its discovery, COVID-19 has rapidly spread across the globe and has had a massive toll on human health, with infection mortality rates as high as 10%, and a crippling impact on the world economy. Despite numerous advances, there remains an urgent need for accurate and rapid point-of-care diagnostic tests and better therapeutic treatment options. To contribute chemically distinct, non-protein-based affinity reagents, we report here the identification of modified DNA-based aptamers that selectively bind to the S1, S2, or receptor-binding domain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein. Several aptamers inhibit the binding of the spike protein to its cell-surface receptor angiotensin-converting enzyme 2 (ACE2) and neutralize authentic SARS-CoV-2 virus in vitro, including all variants of concern. With a high degree of nuclease resistance imparted by the base modifications, these reagents represent a new class of molecules with potential for further development as diagnostics or therapeutics.
ABSTRACT
Many individual genetic risk loci have been associated with multiple common human diseases. However, the molecular basis of this pleiotropy often remains unclear. We present an integrative approach to reveal the molecular mechanism underlying the PROCR locus, associated with lower coronary artery disease (CAD) risk but higher venous thromboembolism (VTE) risk. We identify PROCR-p.Ser219Gly as the likely causal variant at the locus and protein C as a causal factor. Using genetic analyses, human recall-by-genotype and in vitro experimentation, we demonstrate that PROCR-219Gly increases plasma levels of (activated) protein C through endothelial protein C receptor (EPCR) ectodomain shedding in endothelial cells, attenuating leukocyte-endothelial cell adhesion and vascular inflammation. We also associate PROCR-219Gly with an increased pro-thrombotic state via coagulation factor VII, a ligand of EPCR. Our study, which links PROCR-219Gly to CAD through anti-inflammatory mechanisms and to VTE through pro-thrombotic mechanisms, provides a framework to reveal the mechanisms underlying similar cross-phenotype associations.
Subject(s)
Thrombosis , Venous Thromboembolism , Antigens, CD/genetics , Crosses, Genetic , Endothelial Cells/metabolism , Endothelial Protein C Receptor/genetics , Humans , Protein C/metabolism , Receptors, Cell Surface/genetics , Thrombosis/genetics , Venous Thromboembolism/geneticsABSTRACT
PURPOSE: Rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisolone (R-CHOP) represents the standard of care for first-line treatment of diffuse large B-cell lymphoma (DLBCL). However, many patients are unable to tolerate R-CHOP and have inferior outcomes. This study aimed to develop a practical tool to help physicians identify patients with newly diagnosed DLBCL unlikely to tolerate a full course of R-CHOP. METHODS: We developed a predictive model (Tolerability of R-CHOP in Aggressive Lymphoma [TRAIL]) on the basis of a training data set from the phase III GOYA trial (obinutuzumab with CHOP v R-CHOP in 1L DLBCL) using a composite binary end point, identifying patients who prematurely stopped or required reductions of R-CHOP. Candidate predictive variables were selected on the basis of known baseline characteristics that contribute to patient frailty, comorbidity, and/or chemotherapy toxicity. TRAIL was developed using an iterative trial-and-error modeling process to fit a logistic regression model. The final model was evaluated for robustness using a GOYA holdout data set and the phase III MAIN (R-CHOP with or without bevacizumab in 1L DLBCL) R-CHOP-21 data set as external validation. RESULTS: TRAIL includes four simple predictors available in the routine clinical setting: Charlson Comorbidity Index, presence of cardiovascular disease or diabetes, serum albumin, and creatinine clearance. Model generalization performance estimated by the area under the curve was around or above 0.70 across GOYA training, GOYA holdout, and MAIN data sets. Classifying patients into low-, intermediate- and high-risk categories, the proportion of patients experiencing a tolerability event was 3.3%, 12.4%, and 32.9%, respectively, in GOYA holdout, and 9.7%, 9.7%, and 34.2%, respectively, in MAIN. CONCLUSION: TRAIL may be useful as a clinical decision support tool for treatment decisions in patients with DLBCL who may not tolerate standard chemoimmunotherapies.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols , Lymphoma, Large B-Cell, Diffuse , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Clinical Trials, Phase III as Topic , Cyclophosphamide/therapeutic use , Doxorubicin/therapeutic use , Humans , Lymphoma, Large B-Cell, Diffuse/drug therapy , Prednisone/therapeutic use , Rituximab/therapeutic use , Vincristine/therapeutic useABSTRACT
Alveolar macrophages (AMs) and epithelial cells (ECs) are the lone resident lung cells positioned to respond to pathogens at early stages of infection. Extracellular vesicles (EVs) are important vectors of paracrine signaling implicated in a range of (patho)physiologic contexts. Here we demonstrate that AMs, but not ECs, constitutively secrete paracrine activity localized to EVs which inhibits influenza infection of ECs in vitro and in vivo. AMs exposed to cigarette smoke extract lost the inhibitory activity of their secreted EVs. Influenza strains varied in their susceptibility to inhibition by AM-EVs. Only those exhibiting early endosomal escape and high pH of fusion were inhibited via a reduction in endosomal pH. By contrast, strains exhibiting later endosomal escape and lower fusion pH proved resistant to inhibition. These results extend our understanding of how resident AMs participate in host defense and have broader implications in the defense and treatment of pathogens internalized within endosomes.
Subject(s)
Endosomes , Extracellular Vesicles/immunology , Influenza A virus/immunology , Macrophages, Alveolar/immunology , Paracrine Communication/immunology , Virus Internalization , A549 Cells , Animals , Dogs , Endosomes/immunology , Endosomes/pathology , Endosomes/virology , HEK293 Cells , Humans , Macrophages, Alveolar/pathology , Madin Darby Canine Kidney Cells , Mice , Rats , Rats, Wistar , THP-1 CellsABSTRACT
Lung cancer remains the leading cause of cancer-related death in the United States. Although the alveolar macrophage (AM) comprises the major resident immune cell in the lung, few studies have investigated its role in lung cancer development. We recently discovered a potentially novel mechanism wherein AMs regulate STAT-induced inflammatory responses in neighboring epithelial cells (ECs) via secretion and delivery of suppressors of cytokine signaling 3 (SOCS3) within extracellular vesicles (EVs). Here, we explored the impact of SOCS3 transfer on EC tumorigenesis and the integrity of AM SOCS3 secretion during development of lung cancer. AM-derived EVs containing SOCS3 inhibited STAT3 activation as well as proliferation and survival of lung adenocarcinoma cells. Levels of secreted SOCS3 were diminished in lungs of patients with non-small cell lung cancer and in a mouse model of lung cancer, and the impaired ability of murine AMs to secrete SOCS3 within EVs preceded the development of lung tumors. Loss of this homeostatic brake on tumorigenesis prompted our effort to "rescue" it. Provision of recombinant SOCS3 loaded within synthetic liposomes inhibited proliferation and survival of lung adenocarcinoma cells in vitro as well as malignant transformation of normal ECs. Intratumoral injection of SOCS3 liposomes attenuated tumor growth in a lung cancer xenograft model. This work identifies AM-derived vesicular SOCS3 as an endogenous antitumor mechanism that is disrupted within the tumor microenvironment and whose rescue by synthetic liposomes can be leveraged as a potential therapeutic strategy for lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung/immunology , Lung Neoplasms/immunology , Macrophages, Alveolar/immunology , Suppressor of Cytokine Signaling 3 Protein/metabolism , A549 Cells , Alveolar Epithelial Cells/cytology , Alveolar Epithelial Cells/metabolism , Animals , Bronchoalveolar Lavage Fluid/cytology , Bronchoalveolar Lavage Fluid/immunology , Carcinogenesis/drug effects , Carcinogenesis/immunology , Carcinogenesis/pathology , Carcinoma, Non-Small-Cell Lung/drug therapy , Carcinoma, Non-Small-Cell Lung/pathology , Extracellular Vesicles/immunology , Extracellular Vesicles/metabolism , Female , Humans , Injections, Intralesional , Liposomes , Lung Neoplasms/drug therapy , Lung Neoplasms/pathology , Macrophages, Alveolar/cytology , Macrophages, Alveolar/metabolism , Mice , Primary Cell Culture , Rats , Recombinant Proteins/administration & dosage , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , STAT3 Transcription Factor/immunology , STAT3 Transcription Factor/metabolism , Suppressor of Cytokine Signaling 3 Protein/administration & dosage , Suppressor of Cytokine Signaling 3 Protein/genetics , Tumor Microenvironment/drug effects , Tumor Microenvironment/immunology , Xenograft Model Antitumor AssaysABSTRACT
Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.
Subject(s)
Alveolar Epithelial Cells/physiology , Cell Communication/physiology , Cell-Derived Microparticles/physiology , Actins/metabolism , Acute Lung Injury/physiopathology , Animals , Cell Line , Dynamins/metabolism , Endocytosis , Female , Ligands , Macrophages, Alveolar/physiology , Models, Biological , Oxidation-Reduction , Rats , Rats, Wistar , Receptors, Cell Surface/metabolism , Signal Transduction , Smoke/adverse effects , Nicotiana/toxicityABSTRACT
The addition of novel side chains at the 5-position of uracil is an effective means to increase chemical diversity of aptamers and hence the success rate for discovery of high-affinity ligands to protein targets. Such modifications also increase nuclease resistance, which is useful in a range of applications, especially for therapeutics. In this study, we assess the impact of these side chains on plasma pharmacokinetics of modified aptamers conjugated to a 40 kDa polyethylene glycol. We show that clearance from plasma depends on relative hydrophobicity: side chains with a negative cLogP (more hydrophilic) result in slower plasma clearance compared with side chains with a positive cLogP (more hydrophobic). We show that clearance increases with the number of side chains in sequences of ≥28 synthons, but this effect is dramatically diminished in shorter sequences. These results serve as a guide for the design of new therapeutic aptamers with diversity-enhancing side chains.
Subject(s)
Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/pharmacokinetics , Polyethylene Glycols/chemistry , Uracil/chemistry , Animals , Aptamers, Nucleotide/administration & dosage , Aptamers, Nucleotide/blood , Base Sequence , Drug Design , Hydrophobic and Hydrophilic Interactions , Ligands , Linear Models , Male , Polyethylene Glycols/metabolism , Rats , Rats, Sprague-Dawley , SELEX Aptamer Technique/methods , Statistics, Nonparametric , Uracil/metabolismABSTRACT
BACKGROUND: Oxaliplatin in combination with either 5-fluorouracil or capecitabine is commonly used as first-line therapy for patients with small bowel adenocarcinoma. The addition of irinotecan improves survival in other gastrointestinal tumors but at the cost of hematologic toxicity. The authors performed a phase 2 cooperative group study (North Central Cancer Treatment Group N0543, Alliance) using genotype-dosed capecitabine, irinotecan, and oxaliplatin (gCAPIRINOX), with dosing assigned based on UDP glucuronosyltransferase family 1 member A1 (UGT1A1) genotype to test: 1) whether the addition of irinotecan would improve outcomes; and 2) whether UGT1A1 genotype-based dosing could optimize tolerability. METHODS: Previously untreated patients with advanced small bowel adenocarcinoma received irinotecan (day 1), oxaliplatin (day 1), and capecitabine (days 2-15) in a 21-day cycle and were dosed with gCAPIRINOX according to UGT1A1*28 genotypes (6/6, 6/7, and 7/7). RESULTS: A total of 33 patients (17 with the 6/6 genotype, 10 with the 6/7 genotype, and 6 with the 7/7 genotype) were enrolled from October 2007 to November 2013; 73% were male, with a mean age of 64 years (range, 41-77 years). Location of the primary tumor included the duodenum (58%), jejunum (30%), and ileum (9%). The regimen yielded a confirmed response rate of 37.5% (95% confidence interval, 21%-56%), with a median progression-free survival of 8.9 months and a median overall survival of 13.4 months. Neither hematologic toxicity (grade ≥3 in 52.9%, 30.0%, and 33.3%, respectively, of the 6/6, 6/7, and 7/7 genotype groups) nor tumor response rate (41.2%, 33%, and 33%, respectively) were found to differ significantly by UGT1A1 genotype. CONCLUSIONS: UGT1A1 genotype-directed dosing (gCAPIRINOX) appears to be feasible with favorable rates of hematologic toxicity compared with prior 3-drug studies in unselected patients. Larger studies would be needed to determine the regimen's comparability to oxaliplatin and capecitabine (CapeOx) alone or if response/toxicity differs among patients with different UGT1A1 genotypes. Cancer 2017;123:3494-501. © 2017 American Cancer Society.
Subject(s)
Adenocarcinoma/drug therapy , Antineoplastic Combined Chemotherapy Protocols/administration & dosage , Antineoplastic Combined Chemotherapy Protocols/adverse effects , Intestinal Neoplasms/drug therapy , Intestine, Small/pathology , Adenocarcinoma/mortality , Adenocarcinoma/pathology , Adult , Aged , Camptothecin/adverse effects , Camptothecin/analogs & derivatives , Camptothecin/therapeutic use , Cancer Care Facilities , Capecitabine/adverse effects , Capecitabine/therapeutic use , Databases, Factual , Disease-Free Survival , Dose-Response Relationship, Drug , Drug Administration Schedule , Female , Genotype , Glucuronosyltransferase/genetics , Humans , Intestinal Neoplasms/mortality , Intestinal Neoplasms/pathology , Intestine, Small/drug effects , Irinotecan , Kaplan-Meier Estimate , Male , Maximum Tolerated Dose , Middle Aged , Neoplasm Invasiveness/pathology , Neoplasm Staging , Organoplatinum Compounds/adverse effects , Organoplatinum Compounds/therapeutic use , Oxaliplatin , Pharmacogenetics , Prognosis , Proportional Hazards Models , Retrospective Studies , Survival AnalysisABSTRACT
The nucleobases comprising DNA and RNA aptamers provide considerably less chemical diversity than protein-based ligands, limiting their versatility. The introduction of novel functional groups at just one of the four bases in modified aptamers has recently led to dramatic improvement in the success rate of identifying nucleic acid ligands to protein targets. Here we explore the benefits of additional enhancement in physicochemical diversity by selecting modified DNA aptamers that contain amino-acid-like modifications on both pyrimidine bases. Using proprotein convertase subtilisin/kexin type 9 as a representative protein target, we identify specific pairwise combinations of modifications that result in higher affinity, metabolic stability, and inhibitory potency compared with aptamers with single modifications. Such doubly modified aptamers are also more likely to be encoded in shorter sequences and occupy nonoverlapping epitopes more frequently than aptamers with single modifications. These highly modified DNA aptamers have broad utility in research, diagnostic, and therapeutic applications.
Subject(s)
Aptamers, Nucleotide , SELEX Aptamer Technique , Cell Line, Tumor , Deoxyribonucleases/metabolism , Gene Library , Humans , Ligands , PCSK9 Inhibitors , Proprotein Convertase 9/chemistry , Proprotein Convertase 9/geneticsABSTRACT
Unconventional secretion and subsequent uptake of molecular cargo via extracellular vesicles (EVs) is an important mechanism by which cells can exert paracrine effects. While this phenomenon has been widely characterized in the context of their ability to promote inflammation, less is known about the ability of EVs to transfer immunosuppressive cargo. Maintenance of normal physiology in the lung requires suppression of potentially damaging inflammatory responses to the myriad of insults to which it is continually exposed. Recently, our laboratory has reported the ability of alveolar macrophages (AMs) to secrete suppressors of cytokine signaling (SOCS) proteins within microvesicles (MVs) and exosomes (Exos). Uptake of these EVs by alveolar epithelial cells (AECs) resulted in inhibition of pro-inflammatory STAT activation in response to cytokines. Moreover, AM packaging of SOCS within EVs could be rapidly tuned in response to exogenous or AEC-derived substances. In this article we will highlight gaps in knowledge regarding microenvironmental modulation of cargo packaging and utilization as well as EV secretion and uptake. Advances in these areas are critical for improving understanding of intercellular communication in the immune system and for therapeutic application of artificial vesicles aimed at treatment of diseases characterized by dysregulated inflammation.
ABSTRACT
Interleukin-6 (IL-6) is a potent mediator of inflammatory and immune responses, and a validated target for therapeutic intervention of inflammatory diseases. Previous studies have shown that SL1026, a slow off-rate modified aptamer (SOMAmer) antagonist of IL-6, neutralizes IL-6 signaling in vitro. In the present study, we show that SL1026 delays the onset and reduces the severity of rheumatoid symptoms in a collagen-induced arthritis model in cynomolgus monkeys. SL1026 (1 and 10 mg/kg), administered q.i.d., delayed the progression of arthritis and the concomitant increase in serum IL-6 levels compared to the untreated control group. Furthermore, SL1026 inhibited IL-6-induced STAT3 phosphorylation ex vivo in T lymphocytes from human blood and IL-6-induced C-reactive protein and serum amyloid A production in human primary hepatocytes. Importantly, SOMAmer treatment did not elicit an immune response, as evidenced by the absence of anti-SOMAmer antibodies in plasma of treated monkeys. These results demonstrate that SOMAmer antagonists of IL-6 may be attractive agents for the treatment of IL-6-mediated diseases, including rheumatoid arthritis.
Subject(s)
Aptamers, Peptide/chemistry , Arthritis, Experimental/prevention & control , Collagen/adverse effects , Interleukin-6/blood , Amino Acid Sequence , Animals , Arthritis, Experimental/chemically induced , Cells, Cultured , Female , Humans , Interleukin-6/chemistry , Macaca fascicularis , Molecular Sequence Data , Phosphorylation , STAT3 Transcription Factor/metabolism , Sequence Homology, Amino Acid , T-Lymphocytes/metabolismABSTRACT
Discerning the structural building blocks of macromolecules is essential for understanding their folding and function. For a new generation of modified nucleic acid ligands (called slow off-rate modified aptamers or SOMAmers), we previously observed essential functions of hydrophobic aromatic side chains in the context of well-known nucleic acid motifs. Here we report a 2.45-Å resolution crystal structure of a SOMAmer complexed with nerve growth factor that lacks any known nucleic acid motifs, instead adopting a configuration akin to a triangular prism. The SOMAmer utilizes extensive hydrophobic stacking interactions, non-canonical base pairing and irregular purine glycosidic bond angles to adopt a completely non-helical, compact S-shaped structure. Aromatic side chains contribute to folding by creating an unprecedented intercalating zipper-like motif and a prominent hydrophobic core. The structure provides compelling rationale for potent inhibitory activity of the SOMAmer and adds entirely novel motifs to the repertoire of structural elements uniquely available to SOMAmers.
Subject(s)
DNA/chemistry , Nerve Growth Factor/chemistry , Amino Acid Sequence , Base Sequence , Binding Sites , Crystallography, X-Ray , Humans , Hydrophobic and Hydrophilic Interactions , Molecular Sequence Data , Nerve Growth Factor/physiology , Protein Binding , Protein Structure, Secondary , SELEX Aptamer TechniqueABSTRACT
JAK-STAT signaling mediates the actions of numerous cytokines and growth factors, and its endogenous brake is the family of SOCS proteins. Consistent with their intracellular roles, SOCS proteins have never been identified in the extracellular space. Here we report that alveolar macrophages can secrete SOCS1 and -3 in exosomes and microparticles, respectively, for uptake by alveolar epithelial cells and subsequent inhibition of STAT activation. Secretion is tunable and occurs both in vitro and in vivo. SOCS secretion into lung lining fluid was diminished by cigarette smoking in humans and mice. Secretion and transcellular delivery of vesicular SOCS proteins thus represent a new model for the control of inflammatory signaling, which is subject to dysregulation during states of inflammation.
Subject(s)
Cell-Derived Microparticles/immunology , Epithelial Cells/immunology , Macrophages/immunology , Pulmonary Alveoli/immunology , Signal Transduction/immunology , Suppressor of Cytokine Signaling Proteins/immunology , Animals , Cell Line, Transformed , Cell-Derived Microparticles/pathology , Epithelial Cells/pathology , Female , Humans , Inflammation/immunology , Inflammation/pathology , Janus Kinases/immunology , Male , Mice , Pulmonary Alveoli/pathology , Rats , Rats, Wistar , STAT Transcription Factors/immunologyABSTRACT
Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the "diversity gap" between nucleic acid-based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics.
ABSTRACT
Interleukin-6 (IL-6) is a pleiotropic cytokine that regulates immune and inflammatory responses, and its overproduction is a hallmark of inflammatory diseases. Inhibition of IL-6 signaling with the anti-IL-6 receptor antibody tocilizumab has provided some clinical benefit to patients; however, direct cytokine inhibition may be a more effective option. We used the systematic evolution of ligands by exponential enrichment (SELEX) process to discover slow off-rate modified aptamers (SOMAmers) with hydrophobic base modifications that inhibit IL-6 signaling in vitro. Two classes of IL-6 SOMAmers were isolated from modified DNA libraries containing 40 random positions and either 5-(N-benzylcarboxamide)-2'-deoxyuridine (Bn-dU) or 5-[N-(1-naphthylmethyl)carboxamide]-2'-deoxyuridine (Nap-dU) replacing dT. These modifications facilitate the high affinity binding interaction with IL-6 and provide resistance against degradation by serum endonucleases. Post-SELEX optimization of one Bn-dU and one Nap-dU SOMAmer led to improvements in IL-6 binding (10-fold) and inhibition activity (greater than 20-fold), resulting in lead SOMAmers with sub-nanomolar affinity (Kd = 0.2 nm) and potency (IC50 = 0.2 nm). Although similar in inhibition properties, the two SOMAmers have unique sequences and different ortholog specificities. Furthermore, these SOMAmers were stable in human serum in vitro for more than 48 h. Both SOMAmers prevented IL-6 signaling by blocking the interaction of IL-6 with its receptor and inhibited the proliferation of tumor cells in vitro as effectively as tocilizumab. This new class of IL-6 inhibitor may be an effective therapeutic alternative for patients suffering from inflammatory diseases.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Aptamers, Nucleotide/pharmacology , Interleukin-6/antagonists & inhibitors , Interleukin-6/immunology , Receptors, Interleukin-6/immunology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Base Sequence , CHO Cells , Cricetulus , Drug Discovery , Humans , Interleukin-6/chemistry , Interleukin-6/metabolism , Macaca fascicularis , Mice , Molecular Sequence Data , Rats , SELEX Aptamer Technique/methods , Serum/metabolismABSTRACT
Inhalation of ozone (O3), a common environmental pollutant, causes pulmonary injury, pulmonary inflammation, and airway hyperresponsiveness (AHR) in healthy individuals and exacerbates many of these same sequelae in individuals with preexisting lung disease. However, the mechanisms underlying these phenomena are poorly understood. Consequently, we sought to determine the contribution of osteopontin (OPN), a hormone and a pleiotropic cytokine, to the development of O3-induced pulmonary injury, pulmonary inflammation, and AHR. To that end, we examined indices of these aforementioned sequelae in mice genetically deficient in OPN and in wild-type, C57BL/6 mice 24 h following the cessation of an acute (3 h) exposure to filtered room air (air) or O3 (2 parts/million). In wild-type mice, O3 exposure increased bronchoalveolar lavage fluid (BALF) OPN, whereas immunohistochemical analysis demonstrated that there were no differences in the number of OPN-positive alveolar macrophages between air- and O3-exposed wild-type mice. O3 exposure also increased BALF epithelial cells, protein, and neutrophils in wild-type and OPN-deficient mice compared with genotype-matched, air-exposed controls. However, following O3 exposure, BALF neutrophils were significantly reduced in OPN-deficient compared with wild-type mice. When airway responsiveness to inhaled acetyl-ß-methylcholine chloride (methacholine) was assessed using the forced oscillation technique, O3 exposure caused hyperresponsiveness to methacholine in the airways and lung parenchyma of wild-type mice, but not OPN-deficient mice. These results demonstrate that OPN is increased in the air spaces following acute exposure to O3 and functionally contributes to the development of O3-induced pulmonary inflammation and airway and lung parenchymal hyperresponsiveness to methacholine.