ABSTRACT
Challenges in obtaining efficient transduction of brain and spinal cord following systemic AAV delivery have led to alternative administration routes being used in clinical trials that directly infuse the virus into the CNS. However, data comparing different direct AAV injections into the brain remain limited making it difficult to choose optimal routes. Here we tested both AAV9-egfp and AAV9-fLuc delivery via intrastriatal (IST), intracisterna magna (ICM) and lumbar intrathecal (LIT) routes in adult rats and assessed vector distribution and transduction in brain, spinal cord and peripheral tissues. We find that IST infusion leads to robust transgene expression in the striatum, thalamus and cortex with lower peripheral tissue transduction and anti-AAV9 capsid titers compared to ICM or LIT. ICM delivery provided strong GFP and luciferase expression across more brain regions than the other routes and similar expression in the spinal cord to LIT injections, which itself largely failed to transduce the rat brain. Our data highlight the strengths and weaknesses of each direct CNS delivery route which will help with future clinical targeting.
Subject(s)
Gene Transfer Techniques , Spinal Cord , Rats , Animals , Transduction, Genetic , Spinal Cord/metabolism , Brain/metabolism , Transgenes , Genetic Vectors/genetics , Dependovirus/genetics , Dependovirus/metabolismABSTRACT
Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.
Subject(s)
Autoantibodies , Idiopathic Pulmonary Fibrosis , Humans , Idiopathic Pulmonary Fibrosis/drug therapy , Lung/metabolism , Disease Progression , Fibroblasts/metabolismABSTRACT
AIM: Migraine pain is thought to result from activation of meningeal nociceptors that might involve dural mast cell degranulation and release of proteases and pronociceptive mediators. Tryptase, the most abundant dural mast cell protease, has been demonstrated to stimulate dural mast cells, as well as trigeminal nociceptors by activating the protease activated receptor 2. Mast cell or neuronal protease activated receptors 2 may therefore represent a novel target for migraine treatment. In this study, we characterized and evaluated a novel protease activated receptor 2 monoclonal antibody as a preventive anti-migraine pain therapy in preclinical models. METHODS: Flow cytometry, immunocytochemistry, calcium imaging, Homogeneous Time Resolved Technology (HTRF) epitope competition assay and serum pharmacokinetic (PK) assay in rats were performed to confirm the activity, specificity and in vivo stability of PAR650097, a novel anti- protease activated receptor 2 monoclonal antibody. In vivo assessment was performed in female C57BL/6J mice by evaluation of PAR650097 in preventing cutaneous allodynia elicited by (a) supradural injection of the protease activated receptor 2 agonist, Ser-Leu-Ile-Gly-Arg-Leu-amide trifluoroacetate (SLIGRL), or calcitonin gene-related (CGRP) peptide, and (b) induction of latent sensitization by priming with three daily episodes of restraint stress followed by challenge with a subthreshold inhalational exposure to umbellulone (UMB), a transient receptor potential ankyrin 1 (TRPA1) agonist. PAR650097 was administered as a pretreatment prior to the first restraint stress, umbellulone exposure, SLIGRL or calcitonin gene-related peptide injection. Additionally, fremanezumab, a calcitonin gene-related peptide antibody was administered as pre-treatment prior to supradural administration of calcitonin gene-related peptide or SLIGRL. RESULTS: In vitro, PAR650097 demonstrated rapid interaction with protease activated receptor 2, enabling it to fully inhibit protease-induced protease activated receptor 2 activation, in human and mouse cells, with high potency. Furthermore, PAR650097 was highly selective for protease activated receptor 2, demonstrating no affinity for protease activated receptor 1 protein and no functional effect on the activation of cellular protease activated receptor 1 with thrombin. In addition, PAR650097 had an acceptable PK profile, compatible with testing the effects of selective protease activated receptor 2 inhibition in vivo. In vivo, PAR650097 blocked cutaneous allodynia induced by either supradural SLIGRL or calcitonin gene-related peptide. Fremanezumab abolished cutaneous allodynia induced by supradural CGRP, and partially attenuated cutaneous allodynia induced by SLIGRL. Administration of PAR650097, before the first restraint stress episode, did not prevent the acute stress-induced cutaneous allodynia or restraint stress priming revealed by cutaneous allodynia induced by inhalational umbellulone. In contrast, PAR650097 prevented expression of cutaneous allodynia when given before the umbellulone challenge in restraint stress-primed animals. CONCLUSION: PAR650097 specifically inhibits endogenously expressed protease activated receptor 2 in human and mouse cells with high potency. This antibody has an acceptable PK profile in rodents and effectively blocked SLIGR-induced cutaneous allodynia. PAR650097 additionally prevented cutaneous allodynia induced by supradural calcitonin gene-related peptide, indicating that the protease activated receptor 2 receptor is a downstream consequence of calcitonin gene-related peptide actions. Fremanezumab effectively blocked calcitonin gene-related peptide-induced cutaneous allodynia and only partially reduced cutaneous allodynia induced by a protease activated receptor 2 activator, suggesting both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine pain. While PAR650097 did not prevent stress-induced cutaneous allodynia or priming, it effectively prevented cutaneous allodynia induced by a TRPA1 agonist in animals with latent sensitization. Activation of protease activated receptor 2, therefore, contributes to both calcitonin gene-related peptide-dependent and -independent mechanisms in promoting migraine-like pain. Therapeutic targeting of protease activated receptor 2 receptors may represent an anti-migraine pain strategy with a potentially broad efficacy profile.
Subject(s)
Calcitonin Gene-Related Peptide , Migraine Disorders , Animals , Antibodies, Monoclonal , Female , Hyperalgesia/prevention & control , Mice , Mice, Inbred C57BL , Migraine Disorders/drug therapy , Migraine Disorders/prevention & control , Pain , Peptide Hydrolases , Rats , Receptor, PAR-1 , Receptor, PAR-2ABSTRACT
There are no approved drug therapies that can prevent or slow the progression of Parkinson's disease (PD). Accumulation and aggregation of α-synuclein protein is observed throughout the nervous system in PD. α-Synuclein is a core component of Lewy bodies and neurites that neuropathologically define PD, suggesting that α-synuclein may be a key causative agent in PD. Recent experimental data suggest that PD progression may arise due to spreading of pathological forms of extracellular α-synuclein throughout the brain via a cellular release, uptake and seeding mechanism. We have developed a high affinity α-synuclein antibody, MEDI1341, that can enter the brain, sequester extracellular α-synuclein and attenuate α-synuclein spreading in vivo. MEDI1341 binds both monomeric and aggregated forms of α-synuclein. In vitro, MEDI1341 blocks cell-to-cell transmission of pathologically relevant α-synuclein preformed fibrils (pffs). After intravenous injection into rats and cynomolgus monkeys, MEDI1341 rapidly enters the central nervous system and lowers free extracellular α-synuclein levels in the interstitial fluid (ISF) and cerebrospinal fluid (CSF) compartments. Using a novel lentiviral-based in vivo mouse model of α-synuclein spreading in the brain, we show that treatment with MEDI1341 significantly reduces α-synuclein accumulation and propagation along axons. In this same model, we demonstrate that an effector-null version of the antibody was equally as effective as one with effector function. MEDI1341 is now in Phase 1 human clinical trial testing as a novel treatment for α-synucleinopathies including PD with the aim to slow or halt disease progression.
Subject(s)
Antibodies, Monoclonal/pharmacology , Brain/drug effects , alpha-Synuclein/antagonists & inhibitors , Animals , Antibody Specificity , Humans , Macaca fascicularis , Mice , RatsABSTRACT
Previous research has consistently shown that juvenile violent victimization is associated with an increased risk of future criminal involvement, a phenomenon commonly known as victim-offender overlap. Despite a growing interest in the factors underlying this overlap, potential roles of major life transitions and turning points that may interrupt and reshape the nature of this developmental association have garnered less academic attention. Analyzing nationally representative data from waves I, IV, and V of the National Longitudinal Study of Adolescent to Adult Health (Add Health; n = 10,205), this study investigates the association between juvenile violent victimization and adult criminal outcomes (i.e., violent offending, non-violent offending, arrest, and incarceration) and whether this association is moderated by military service with and without combat experience in young adulthood. Employing a series of logistic regression analyses and adjusting for a host of covariates, measures of selection, and criminogenic traits, we found that juvenile violent victimization was significantly associated with greater odds of violent offending, arrest, and incarceration in adulthood. Among individuals with violent victimization histories, military service with no combat experience was associated with a 16% decrease in the odds of incarceration in adulthood. Combat experience, however, was associated with over seven times greater odds of violent offending in adulthood for these individuals. These findings have important implications for theory, research, and practice, and highlight the relevance of life transitions and turning points in general, and military service in particular, in mitigating or perpetuating the criminogenic impacts of violent victimization in the life course.
Subject(s)
Crime Victims , Criminals , Military Personnel , Adult , Adolescent , Humans , Young Adult , Violence , Longitudinal StudiesABSTRACT
Native mass spectrometry is a potent method for characterizing biomacromolecular assemblies. A critical aspect to extracting accurate mass information is the correct inference of the ion ensemble charge states. While a variety of experimental strategies and algorithms have been developed to facilitate this, virtually all approaches rely on the implicit assumption that any peaks in a native mass spectrum can be directly attributed to an underlying charge state distribution. Here, we demonstrate that this paradigm breaks down for several types of macromolecular protein complexes due to the intrinsic heterogeneity induced by the stochastic nature of their assembly. Utilizing several protein assemblies of adeno-associated virus capsids and ferritin, we demonstrate that these particles can produce a variety of unexpected spectral appearances, some of which appear superficially similar to a resolved charge state distribution. When interpreted using conventional charge inference strategies, these distorted spectra can lead to substantial errors in the calculated mass (up to â¼5%). We provide a novel analytical framework to interpret and extract mass information from these spectra by combining high-resolution native mass spectrometry, single particle Orbitrap-based charge detection mass spectrometry, and sophisticated spectral simulations based on a stochastic assembly model. We uncover that these mass spectra are extremely sensitive to not only mass heterogeneity within the subunits, but also to the magnitude and width of their charge state distributions. As we postulate that many protein complexes assemble stochastically, this framework provides a generalizable solution, further extending the usability of native mass spectrometry in the characterization of biomacromolecular assemblies.
ABSTRACT
Preclinical studies of PD-L1 and CTLA-4 blockade have relied heavily on mouse syngeneic tumor models with intact immune systems, which facilitate dissection of immunosuppressive mechanisms in the tumor microenvironment. Commercially developed monoclonal antibodies (mAbs) targeting human PD-L1, PD-1, and CTLA-4 may not demonstrate cross-reactive binding to their mouse orthologs, and surrogate anti-mouse antibodies are often used in their place to inhibit these immune checkpoints. In each case, multiple choices exist for surrogate antibodies, which differ with respect to species of origin, affinity, and effector function. To develop relevant murine surrogate antibodies for the anti-human PD-L1 mAb durvalumab and the anti-human CTLA-4 mAb tremelimumab, rat/mouse chimeric or fully murine mAbs engineered for reduced effector function were developed and compared with durvalumab and tremelimumab. Characterization included determination of target affinity, in vivo effector function, pharmacokinetic profile, and anti-tumor efficacy in mouse syngeneic tumor models. Results showed that anti-PD-L1 and anti-CTLA-4 murine surrogates with pharmacologic properties similar to those of durvalumab and tremelimumab demonstrated anti-tumor activity in a subset of commonly used mouse syngeneic tumor models. This activity was not entirely dependent on antibody-dependent cellular cytotoxicity, antibody-dependent cellular phagocytosis effector function, or regulatory T-cell depletion, as antibodies engineered to lack these features showed activity in models historically sensitive to checkpoint inhibition, albeit at a significantly lower level than antibodies with intact effector function.
Subject(s)
Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Monoclonal/therapeutic use , Neoplasms, Experimental/drug therapy , T-Lymphocytes, Regulatory/drug effects , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized/immunology , Antineoplastic Agents, Immunological/immunology , Antineoplastic Agents, Immunological/therapeutic use , B7-H1 Antigen/immunology , CTLA-4 Antigen/immunology , Cell Line, Tumor , Female , Humans , Kaplan-Meier Estimate , Mice, Inbred BALB C , Mice, Inbred C57BL , Neoplasms, Experimental/immunology , Neoplasms, Experimental/pathology , Rats, Sprague-Dawley , T-Lymphocytes, Regulatory/immunology , Tumor Burden/drug effects , Tumor Burden/immunologyABSTRACT
The recombinant adeno-associated virus (AAV) vector is one of the most utilized viral vectors in gene therapy due to its robust, long-term in vivo transgene expression and low toxicity. One major hurdle for clinical AAV applications is large-scale manufacturing. In this regard, the baculovirus-based AAV production system is highly attractive due to its scalability and predictable biosafety. Here, we describe a simple method to improve the baculovirus-based AAV production using the ExpiSf Baculovirus Expression System with a chemically defined medium for suspension culture of high-density ExpiSf9 cells. Baculovirus-infected ExpiSf9 cells produced up to 5 × 1011 genome copies of highly purified AAV vectors per 1 mL of suspension culture, which is up to a 19-fold higher yield than the titers we obtained from the conventional Sf9 cell-based system. When mice were administered the same dose of AAV vectors, we saw comparable transduction efficiency and biodistributions between the vectors made in ExpiSf9 and Sf9 cells. Thus, the ExpiSf Baculovirus Expression System would support facile and scalable AAV manufacturing amenable for preclinical and clinical applications.
ABSTRACT
Filopodia are finger-like actin-rich protrusions that extend from the cell surface and are important for cell-cell communication and pathogen internalization. The small size and transient nature of filopodia combined with shared usage of actin regulators within cells confounds attempts to identify filopodial proteins. Here, we used phage display phenotypic screening to isolate antibodies that alter the actin morphology of filopodia-like structures (FLS) in vitro. We found that all of the antibodies that cause shorter FLS interact with SNX9, an actin regulator that binds phosphoinositides during endocytosis and at invadopodia. In cells, we discover SNX9 at specialized filopodia in Xenopus development and that SNX9 is an endogenous component of filopodia that are hijacked by Chlamydia entry. We show the use of antibody technology to identify proteins used in filopodia-like structures, and a role for SNX9 in filopodia.
Subject(s)
Pseudopodia/metabolism , Sorting Nexins/metabolism , Xenopus Proteins/metabolism , Animals , Female , HeLa Cells , Humans , Male , Sorting Nexins/genetics , Xenopus Proteins/genetics , Xenopus laevisABSTRACT
Objective: Amyloid-beta oligomers (Aßo) trigger the development of Alzheimer's disease (AD) pathophysiology. Cellular prion protein (PrPC) initiates synaptic damage as a high affinity receptor for Aßo. Here, we evaluated the preclinical therapeutic efficacy of a fully human monoclonal antibody against PrPC. This AZ59 antibody selectively targets the Aßo binding site in the amino-terminal unstructured domain of PrPC to avoid any potential risk of direct toxicity. Methods: Potency of AZ59 was evaluated by binding to PrPC, blockade of Aßo interaction and interruption of Aßo signaling. AZ59 was administered to mice by weekly intraperitoneal dosing and brain antibody measured. APP/PS1 transgenic mice were treated with AZ59 and assessed by memory tests, by brain biochemistry and by histochemistry for Aß, gliosis and synaptic density. Results: AZ59 binds PrPC with 100 pmol/L affinity and blocks human brain Aßo binding to PrPC, as well as prevents synaptotoxic signaling. Weekly i.p. dosing of 20 mg/kg AZ59 in a murine form achieves trough brain antibody levels greater than 10 nmol/L. Aged symptomatic APP/PS1 transgenic mice treated with AZ59 for 5-7 weeks show a full rescue of behavioral and synaptic loss phenotypes. This recovery occurs without clearance of plaque pathology or elimination of gliosis. AZ59 treatment also normalizes synaptic signaling abnormalities in transgenic brain. These benefits are dose-dependent and persist for at least 1 month after the last dose. Interpretation: Preclinical data demonstrate that systemic AZ59 therapy rescues central synapses and memory function from transgenic Alzheimer's disease pathology, supporting a disease-modifying therapeutic potential.
Subject(s)
Alzheimer Disease/drug therapy , Alzheimer Disease/pathology , Antibodies, Monoclonal/therapeutic use , PrPC Proteins/antagonists & inhibitors , PrPC Proteins/immunology , Amyloid beta-Peptides/metabolism , Animals , Binding Sites , Brain/pathology , COS Cells , Chlorocebus aethiops , Cognition , Disease Models, Animal , Humans , MAP Kinase Signaling System , Mice , Mice, Inbred C57BL , Mice, Transgenic , Signal Transduction , Synapses/pathologyABSTRACT
P2X4 is a ligand-gated ion channel implicated in neuropathic pain. Drug discovery efforts targeting P2X4 have been unsuccessful largely because of the difficulty in engineering specificity and selectivity. Here, we describe for the first time the generation of a panel of diverse monoclonal antibodies (mAbs) to human and mouse P2X4, capable of both positive and negative modulation of channel function. The affinity-optimised anti-P2X4 mAb IgG#151-LO showed exquisite selectivity for human P2X4 and induced potent and complete block of P2X4 currents. Site-directed mutagenesis of P2X4 revealed the head domain as a key interaction site for inhibitory mAbs. Inhibition of spinal P2X4 either by intrathecal delivery of an anti-P2X4 mAb or by systemic delivery of an anti-P2X4 bispecific mAb with enhanced blood-spinal cord barrier permeability produced long-lasting (>7 days) analgesia in a mouse model of neuropathic pain. We therefore propose that inhibitory mAbs binding the head domain of P2X4 have therapeutic potential for the treatment of neuropathic pain.
Subject(s)
Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/metabolism , Neuralgia/metabolism , Neuralgia/prevention & control , Receptors, Purinergic P2X4/metabolism , Animals , Cells, Cultured , Female , HEK293 Cells , Humans , Injections, Spinal , Mice , Mice, Inbred C57BL , Protein Binding/physiology , Purinergic P2X Receptor Antagonists/administration & dosage , Purinergic P2X Receptor Antagonists/metabolism , Rats , Rats, Sprague-DawleyABSTRACT
C5a is a potent anaphylatoxin that modulates inflammation through the C5aR1 and C5aR2 receptors. The molecular interactions between C5a-C5aR1 receptor are well defined, whereas C5a-C5aR2 receptor interactions are poorly understood. Here, we describe the generation of a human antibody, MEDI7814, that neutralizes C5a and C5adesArg binding to the C5aR1 and C5aR2 receptors, without affecting complement-mediated bacterial cell killing. Unlike other anti-C5a mAbs described, this antibody has been shown to inhibit the effects of C5a by blocking C5a binding to both C5aR1 and C5aR2 receptors. The crystal structure of the antibody in complex with human C5a reveals a discontinuous epitope of 22 amino acids. This is the first time the epitope for an antibody that blocks C5aR1 and C5aR2 receptors has been described, and this work provides a basis for molecular studies aimed at further understanding the C5a-C5aR2 receptor interaction. MEDI7814 has therapeutic potential for the treatment of acute inflammatory conditions in which both C5a receptors may mediate inflammation, such as sepsis or renal ischemia-reperfusion injury.
Subject(s)
Antibodies, Monoclonal/pharmacology , Antibody Affinity , Complement C5a/antagonists & inhibitors , Receptor, Anaphylatoxin C5a/antagonists & inhibitors , Receptors, Chemokine/antagonists & inhibitors , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/genetics , Antibodies, Monoclonal/immunology , Antibody Specificity , Binding Sites, Antibody , Complement C5a/chemistry , Complement C5a/immunology , Complement C5a/metabolism , Epitope Mapping/methods , Epitopes , HEK293 Cells , Humans , Protein Binding , Protein Conformation , Protein Engineering , Receptor, Anaphylatoxin C5a/chemistry , Receptor, Anaphylatoxin C5a/immunology , Receptor, Anaphylatoxin C5a/metabolism , Receptors, Chemokine/chemistry , Receptors, Chemokine/immunology , Receptors, Chemokine/metabolism , Structure-Activity RelationshipABSTRACT
Calcitonin gene-related peptide (CGRP) is a small neuropeptide and a potent vasodilator that is widely associated with chronic pain and migraine. An antibody that inhibits CGRP function would be a potential therapeutic for treatment of these disorders. Here we describe the isolation of highly potent antibodies to CGRP from phage and ribosome display libraries and characterization of their epitope, species cross-reactivity, kinetics, and functional activity. Homogenous time-resolved fluorescence (HTRF) binding assays identified antibodies with the desired species cross-reactivity from naïve libraries, and HTRF epitope competition assays were used to characterize and group scFv by epitope. The functional inhibition of CGRP and species cross-reactivity of purified scFv and antibodies were subsequently confirmed using cAMP assays. We show that epitope competition assays could be used as a surrogate for functional cell-based assays during affinity maturation, in combination with scFv off-rate ranking by biolayer interferometry (BLI). This is the first time it has been shown that off-rate ranking can be predictive of functional activity for anti-CGRP antibodies. Here we demonstrate how, by using just four simple assays, diverse panels of antibodies to CGRP can be identified. These assay formats have potential utility in the identification of antibodies to other therapeutic targets.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Biological Assay/methods , Calcitonin Gene-Related Peptide/immunology , Cell Line , Epitopes/immunology , Fluorescence , HumansABSTRACT
Uncontrolled self-association is a major challenge in the exploitation of proteins as therapeutics. Here we describe the development of a structural proteomics approach to identify the amino acids responsible for aberrant self-association of monoclonal antibodies and the design of a variant with reduced aggregation and increased serum persistence in vivo. We show that the human monoclonal antibody, MEDI1912, selected against nerve growth factor binds with picomolar affinity, but undergoes reversible self-association and has a poor pharmacokinetic profile in both rat and cynomolgus monkeys. Using hydrogen/deuterium exchange and cross-linking-mass spectrometry we map the residues responsible for self-association of MEDI1912 and show that disruption of the self-interaction interface by three mutations enhances its biophysical properties and serum persistence, whilst maintaining high affinity and potency. Immunohistochemistry suggests that this is achieved via reduction of non-specific tissue binding. The strategy developed represents a powerful and generic approach to improve the properties of therapeutic proteins.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Protein Engineering/methods , Animals , Antibodies, Monoclonal/pharmacokinetics , Biophysical Phenomena , Chromatography, Gel , Enzyme-Linked Immunosorbent Assay , HEK293 Cells , Humans , Hydrogen , Mice , Mutation/genetics , Organ Specificity , Protein Binding , Protein Conformation , Protein Interaction Mapping , Protein Multimerization , Rats , Spectrometry, Mass, Electrospray Ionization , Surface Properties , ViscosityABSTRACT
Anti-idiotype antibodies against a therapeutic antibody are key reagents for the development of immunogenicity and pharmacokinetic (PK) assays during pre-clinical and clinical development. Here we have used a combination of phage and ribosome display to isolate a panel of monoclonal anti-idiotype antibodies with sub-nanomolar affinity and high specificity to a human anti-IgE monoclonal antibody. Anti-idiotype antibodies were enriched from scFv libraries using phage display, and a biochemical epitope competition assay was used to identify anti-idiotypes which neutralized IgE binding, which was essential for the intended use of the anti-idiotypes as positive controls in neutralizing anti-drug antibody (Nab) assays. The phage display-derived anti-idiotype antibodies were rapidly affinity-matured using a random point mutagenesis approach in ribosome display. Ten anti-idiotype antibodies with improved neutralizing activity relative to the parent antibodies displayed sub-nanomolar affinity for the anti-IgE antibody, representing up to 20-fold improvements in affinity from just two rounds of affinity-based selection. The optimized anti-idiotype antibodies retained the specificity of the parent antibodies, and importantly, were fit for purpose for use in PK and anti-drug antibody (ADA) assays. The approach we describe here for generation of anti-idiotype antibodies to an anti-IgE antibody is generically applicable for the rapid isolation and affinity maturation of anti-idiotype antibodies to any antibody-based drug candidate.
Subject(s)
Antibodies, Neutralizing/immunology , Antibody Formation/immunology , Bacteriophages/immunology , Immunoglobulin Idiotypes/immunology , Ribosomes/immunology , Antibodies, Anti-Idiotypic/immunology , Antibodies, Monoclonal/immunology , Antibody Affinity/immunology , Antibody Specificity/immunology , Humans , Peptide LibraryABSTRACT
Immunization of mice or rats with a "non-self" protein is a commonly used method to obtain monoclonal antibodies, and relies on the immune system's ability to recognize the immunogen as foreign. Immunization of an antigen with 100% identity to the endogenous protein, however, will not elicit a robust immune response. To develop antibodies to mouse proteins, we focused on the potential for breaking such immune tolerance by genetically fusing two independent T-cell epitope-containing sequences (from tetanus toxin (TT) and diphtheria toxin fragment A (DTA)) to a mouse protein, mouse ST2 (mST2). Wild-type CD1 mice were immunized with three mST2 tagged proteins (Fc, TT and DTA) and the specific serum response was determined. Only in mice immunized with the T-cell epitope-containing antigens were specific mST2 serum responses detected; hybridomas generated from these mice secreted highly sequence-diverse IgGs that were capable of binding mST2 and inhibiting the interaction of mST2 with its ligand, mouse interleukin (IL)-33 (mIL-33). Of the hundreds of antibodies profiled, we identified five potent antibodies that were able to inhibit IL-33 induced IL-6 release in a mast cell assay; notably one such antibody was sufficiently potent to suppress IL-5 release and eosinophilia infiltration in an Alternaria alternata challenge mouse model of asthma. This study demonstrated, for the first time, that T-cell epitope-containing tags have the ability to break tolerance in wild-type mice to 100% conserved proteins, and it provides a compelling argument for the broader use of this approach to generate antibodies against any mouse protein or conserved ortholog.
Subject(s)
Antibodies, Monoclonal, Murine-Derived/immunology , Antibody Specificity , Epitopes, T-Lymphocyte/immunology , Receptors, Interleukin/immunology , Animals , Antibodies, Monoclonal, Murine-Derived/pharmacology , Asthma/drug therapy , Asthma/immunology , Asthma/pathology , Cell Line, Transformed , Diphtheria Toxin/chemistry , Diphtheria Toxin/immunology , Epitopes, T-Lymphocyte/chemistry , Female , Humans , Interleukin-1 Receptor-Like 1 Protein , Mice , Mice, Inbred BALB C , Rats , Receptors, Interleukin/chemistry , Tetanus Toxin/chemistry , Tetanus Toxin/immunologyABSTRACT
The critical role played by IgE in allergic asthma is well-documented and clinically precedented, but some patients in whom IgE neutralization may still offer clinical benefit are excluded from treatment with the existing anti-IgE therapy, omalizumab, due to high total IgE levels or body mass. In this study, we sought to generate a novel high affinity anti-IgE antibody (MEDI4212) with potential to treat a broad severe asthma patient population. Analysis of body mass, total and allergen-specific IgE levels in a cohort of severe asthmatics was used to support the rationale for development of a high affinity IgE-targeted antibody therapeutic. Phage display technology was used to generate a human IgG1 lead antibody, MEDI4212, which was characterized in vitro using binding, signaling and functional assay systems. Protein crystallography was used to determine the details of the interaction between MEDI4212 and IgE. MEDI4212 bound human IgE with an affinity of 1.95 pM and was shown to target critical residues in the IgE Cε3 domain critical for interaction with FcεRI. MEDI4212 potently inhibited responses through FcεRI and also prevented the binding of IgE to CD23. When used ex vivo at identical concentration, MEDI4212 depleted free-IgE from human sera to levels ~1 log lower than omalizumab. Our results thus indicate that MEDI4212 is a novel, high affinity antibody that binds specifically to IgE and prevents IgE binding to its receptors. MEDI4212 effectively depleted free-IgE from human sera ex vivo to a level (1 IU/mL) anticipated to provide optimal IgE suppression in severe asthma patients.
Subject(s)
Antibodies, Anti-Idiotypic/immunology , Antibodies, Anti-Idiotypic/therapeutic use , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/therapeutic use , Asthma/immunology , Asthma/therapy , Immunoglobulin E/immunology , Adolescent , Adult , Aged , Antibodies, Anti-Idiotypic/genetics , Antibodies, Monoclonal, Humanized/therapeutic use , Antibodies, Neutralizing/genetics , Antibody Affinity , Antibody Specificity , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/immunology , Antigen-Antibody Reactions , Binding Sites , Cohort Studies , Humans , Immunoglobulin E/chemistry , Immunoglobulin E/metabolism , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin G/therapeutic use , Middle Aged , Models, Molecular , Omalizumab , Peptide Library , Receptors, IgE/metabolism , Young AdultABSTRACT
Apoptosis through the TRAIL receptor pathway can be induced via agonistic IgG to either TRAIL-R1 or TRAIL-R2. Here we describe the use of phage display to isolate a substantive panel of fully human anti-TRAIL receptor single chain Fv fragments (scFvs); 234 and 269 different scFvs specific for TRAIL-R1 and TRAIL-R2 respectively. In addition, 134 different scFvs that were cross-reactive for both receptors were isolated. To facilitate screening of all 637 scFvs for potential agonistic activity in vitro, a novel high-throughput surrogate apoptosis assay was developed. Ten TRAIL-R1 specific scFv and 6 TRAIL-R2 specific scFv were shown to inhibit growth of tumor cells in vitro in the absence of any cross-linking agents. These scFv were all highly specific for either TRAIL-R1 or TRAIL-R2, potently inhibited tumor cell proliferation, and were antagonists of TRAIL binding. Moreover, further characterization of TRAIL-R1 agonistic scFv demonstrated significant anti-tumor activity when expressed and purified as a monomeric Fab fragment. Thus, scFv and Fab fragments, in addition to whole IgG, can be agonistic and induce tumor cell death through specific binding to either TRAIL-R1 or TRAIL-R2. These potent agonistic scFv were all isolated directly from the starting phage antibody library and demonstrated significant tumor cell killing properties without any requirement for affinity maturation. Some of these selected scFv have been converted to IgG format and are being studied extensively in clinical trials to investigate their potential utility as human monoclonal antibody therapeutics for the treatment of human cancer.
Subject(s)
Immunoglobulin Fab Fragments/pharmacology , Immunotherapy , Neoplasms/therapy , Receptors, TNF-Related Apoptosis-Inducing Ligand/agonists , Single-Chain Antibodies/pharmacology , Antibody-Dependent Cell Cytotoxicity/genetics , Antibody-Dependent Cell Cytotoxicity/immunology , Apoptosis/drug effects , Apoptosis/immunology , Binding, Competitive , Caspase 3/metabolism , Cell Proliferation/drug effects , Genetic Engineering , HeLa Cells , High-Throughput Screening Assays , Humans , Immunoglobulin Fab Fragments/genetics , Immunoglobulin Fab Fragments/isolation & purification , Immunoglobulin Fab Fragments/metabolism , Neoplasms/immunology , Neoplasms/pathology , Single-Chain Antibodies/biosynthesis , Single-Chain Antibodies/genetics , Single-Chain Antibodies/isolation & purificationABSTRACT
OBJECTIVE: To identify and characterize a fully human antibody directed against B lymphocyte stimulator (BLyS), a tumor necrosis factor-related cytokine that plays a critical role in the regulation of B cell maturation and development. Elevated levels of BLyS have been implicated in the pathogenesis of autoimmune diseases. METHODS: A human phage display library was screened for antibodies against human BLyS. A human monoclonal antibody, LymphoStat-B, specific for human BLyS was obtained from the library screening and subsequent affinity optimization mutagenesis. The antibody was tested for inhibition of human BLyS in vitro and in an in vivo murine model. Additionally, the consequences of BLyS inhibition were tested in vivo by administration of LymphoStat-B to cynomolgus monkeys. RESULTS: LymphoStat-B bound with high affinity to human BLyS and inhibited the binding of BLyS to its 3 receptors, TACI, BCMA, and BLyS receptor 3/BAFF-R. LymphoStat-B potently inhibited BLyS-induced proliferation of B cells in vitro, and administration of LymphoStat-B to mice prevented human BLyS-induced increases in splenic B cell numbers and IgA titers. In cynomolgus monkeys, administration of LymphoStat-B resulted in decreased B cell representation in both spleen and mesenteric lymph nodes. CONCLUSION: A fully human monoclonal antibody has been isolated that binds to BLyS with high affinity and neutralizes human BLyS bioactivity in vitro and in vivo. Administration of this antibody to cynomolgus monkeys resulted in B cell depletion in spleen and lymph node. This antibody may prove therapeutically useful in the treatment of autoimmune diseases in humans.