ABSTRACT
Hypertension remains a leading cause of cardiovascular and kidney diseases. Failure to control blood pressure with ≥ 3 medications or control requiring ≥ 4 medications is classified as resistant hypertension (rHTN) and new therapies are needed to reduce the resulting increased risk of morbidity and mortality. Here, we report genetic evidence that relaxin family peptide receptor 2 (RXFP2) is associated with rHTN in men, but not in women. This study shows that adrenal gland gene expression of RXFP2 is increased in men with hypertension and the RXFP2 natural ligand, INSL3, increases adrenal steroidogenesis and corticosteroid secretion in human adrenal cells. To address the hypothesis that RXFP2 activation is an important mechanism in rHTN, we discovered and characterized small molecule and monoclonal antibody (mAb) blockers of RXFP2. The novel chemical entities and mAbs show potent, selective inhibition of RXFP2 and reduce aldosterone and cortisol synthesis and release. The RXFP2 mAbs have suitable rat pharmacokinetic profiles to evaluate the role of RXFP2 in the development and maintenance of rHTN. Overall, we identified RXFP2 activity as a potential new mechanism in rHTN and discovered RXFP2 antagonists for the future interrogation of RXFP2 in cardiovascular and renal diseases.
Subject(s)
Hypertension , Receptors, G-Protein-Coupled , Receptors, Peptide , Humans , Male , Hypertension/drug therapy , Hypertension/genetics , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Animals , Receptors, Peptide/genetics , Receptors, Peptide/metabolism , Receptors, Peptide/antagonists & inhibitors , Rats , Female , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Adrenal Glands/metabolism , Adrenal Glands/drug effects , Drug Resistance/genetics , Antihypertensive Agents/pharmacology , Aldosterone/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Human CLDN18.2 is highly expressed in a significant proportion of gastric and pancreatic adenocarcinomas, while normal tissue expression is limited to the epithelium of the stomach. The restricted expression makes it a potential drug target for the treatment of gastric and pancreatic adenocarcinoma, as evidenced by efforts to target CLDN18.2 via naked antibody and CAR-T modalities. Herein we describe CLDN18.2-targeting via a CD3-bispecific and an antibody drug conjugate and the characterization of these potential therapeutic molecules in efficacy and preliminary toxicity studies. Anti-hCLDN18.2 ADC, CD3-bispecific and diabody, targeting a protein sequence conserved in rat, mouse and monkey, exhibited in vitro cytotoxicity in BxPC3/hCLDN18.2 (IC50 = 1.52, 2.03, and 0.86 nM) and KATO-III/hCLDN18.2 (IC50 = 1.60, 0.71, and 0.07 nM) respectively and inhibited tumor growth of pancreatic and gastric patient-derived xenograft tumors. In a rat exploratory toxicity study, the ADC was tolerated up to 10 mg/kg. In a preliminary assessment of tolerability, the anti-CLDN18.2 diabody (0.34 mg/kg) did not produce obvious signs of toxicity in the stomach of NSG mice 4 weeks after dosing. Taken together, our data indicate that targeting CLDN18.2 with an ADC or bispecific modality could be a valid therapeutic approach for the treatment of gastric and pancreatic cancer.
Subject(s)
Antibodies, Bispecific/immunology , CD3 Complex/immunology , Claudins/immunology , Immunoconjugates/therapeutic use , Pancreatic Neoplasms/therapy , Stomach Neoplasms/therapy , Adenocarcinoma/therapy , Animals , Carcinoma, Pancreatic Ductal/therapy , Cell Line, Tumor , Humans , Immunoconjugates/blood , Mice , Pancreatic Neoplasms/blood , Rats , Stomach Neoplasms/bloodABSTRACT
Monoclonal antibodies are the largest class of therapeutic proteins due in part to their ability to bind an antigen with a high degree of affinity and specificity. A precise determination of their epitope is important for gaining insights into their therapeutic mechanism of action and to help differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of multiple antibodies in parallel over the course of just several weeks. This approach is based on a combination of rational library design, yeast surface display, and next generation DNA sequencing and provides quantitative insights into the epitope residues most critical for the antibody-antigen interaction. As an example, we will use this method to map the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus.
Subject(s)
Antibodies, Monoclonal/immunology , Epitope Mapping/methods , Epitopes/immunology , High-Throughput Nucleotide Sequencing/methods , Animals , Epitopes/genetics , Humans , Mutagenesis , Peptide Library , Saccharomyces cerevisiae/geneticsABSTRACT
Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.
Subject(s)
Drug Delivery Systems , Immunoconjugates/pharmacology , Lysosomes/metabolism , Transcytosis , Amyloid beta-Protein Precursor/immunology , Amyloid beta-Protein Precursor/therapeutic use , Animals , Antibodies, Bispecific/therapeutic use , Antineoplastic Agents/chemistry , Cell Line, Tumor , Humans , Immunoconjugates/metabolism , Mice, Nude , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/therapeutic use , Receptor, ErbB-2/immunology , Receptor, ErbB-2/therapeutic useABSTRACT
Staphylococcus aureus is both an important pathogen and a human commensal. To explore this ambivalent relationship between host and microbe, we analysed the memory humoral response against IsdB, a protein involved in iron acquisition, in four healthy donors. Here we show that in all donors a heavily biased use of two immunoglobulin heavy chain germlines generated high affinity (pM) antibodies that neutralize the two IsdB NEAT domains, IGHV4-39 for NEAT1 and IGHV1-69 for NEAT2. In contrast to the typical antibody/antigen interactions, the binding is primarily driven by the germline-encoded hydrophobic CDRH-2 motifs of IGHV1-69 and IGHV4-39, with a binding mechanism nearly identical for each antibody derived from different donors. Our results suggest that IGHV1-69 and IGHV4-39, while part of the adaptive immune system, may have evolved under selection pressure to encode a binding motif innately capable of recognizing and neutralizing a structurally conserved protein domain involved in pathogen iron acquisition.
Subject(s)
Antibodies, Bacterial/immunology , Bacterial Proteins/immunology , Staphylococcal Infections/microbiology , Virulence Factors/immunology , Antibodies, Neutralizing , B-Lymphocytes , Humans , Immunologic Memory , Models, Molecular , Protein Conformation , Protein Domains , RNA, Long Noncoding , Staphylococcal Infections/immunology , Staphylococcus aureusABSTRACT
The prominent role of voltage-gated sodium channel 1.7 (Nav1.7) in nociception was revealed by remarkable human clinical and genetic evidence. Development of potent and subtype-selective inhibitors of this ion channel is crucial for obtaining therapeutically useful analgesic compounds. Microproteins isolated from animal venoms have been identified as promising therapeutic leads for ion channels, because they naturally evolved to be potent ion channel blockers. Here, we report the engineering of highly potent and selective inhibitors of the Nav1.7 channel based on tarantula ceratotoxin-1 (CcoTx1). We utilized a combination of directed evolution, saturation mutagenesis, chemical modification, and rational drug design to obtain higher potency and selectivity to the Nav1.7 channel. The resulting microproteins are highly potent (IC50 to Nav1.7 of 2.5 nm) and selective. We achieved 80- and 20-fold selectivity over the closely related Nav1.2 and Nav1.6 channels, respectively, and the IC50 on skeletal (Nav1.4) and cardiac (Nav1.5) sodium channels is above 3000 nm The lead molecules have the potential for future clinical development as novel therapeutics in the treatment of pain.
Subject(s)
NAV1.7 Voltage-Gated Sodium Channel/chemistry , Pain Management/methods , Protein Engineering , Voltage-Gated Sodium Channel Blockers/pharmacology , HEK293 Cells , Humans , NAV1.7 Voltage-Gated Sodium Channel/drug effects , Patch-Clamp Techniques , Phylogeny , Spider Venoms/chemistryABSTRACT
Purinergic homomeric P2X3 and heteromeric P2X2/3 receptors are ligand-gated cation channels activated by ATP. Both receptors are predominantly expressed in nociceptive sensory neurons, and an increase in extracellular ATP concentration under pathological conditions, such as tissue damage or visceral distension, induces channel opening, membrane depolarization, and initiation of pain signaling. Hence, these receptors are considered important therapeutic targets for pain management, and development of selective antagonists is currently progressing. To advance the search for novel analgesics, we have generated a panel of monoclonal antibodies directed against human P2X3 (hP2X3). We have found that these antibodies produce distinct functional effects, depending on the homomeric or heteromeric composition of the target, its kinetic state, and the duration of antibody exposure. The most potent antibody, 12D4, showed an estimated IC50 of 16 nm on hP2X3 after short term exposure (up to 18 min), binding to the inactivated state of the channel to inhibit activity. By contrast, with the same short term application, 12D4 potentiated the slow inactivating current mediated by the heteromeric hP2X2/3 channel. Extending the duration of exposure to â¼20 h resulted in a profound inhibition of both homomeric hP2X3 and heteromeric hP2X2/3 receptors, an effect mediated by efficient antibody-induced internalization of the channel from the plasma membrane. The therapeutic potential of mAb12D4 was assessed in the formalin, complete Freund's adjuvant, and visceral pain models. The efficacy of 12D4 in the visceral hypersensitivity model indicates that antibodies against P2X3 may have therapeutic potential in visceral pain indications.
Subject(s)
Antibodies, Monoclonal/pharmacology , Purinergic P2X Receptor Antagonists/pharmacology , Receptors, Purinergic P2X2/immunology , Receptors, Purinergic P2X3/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity/immunology , Cell Line, Tumor , Cells, Cultured , Female , Freund's Adjuvant , HEK293 Cells , Humans , Inflammation/chemically induced , Inflammation/metabolism , Inflammation/prevention & control , Ion Channels/chemistry , Ion Channels/metabolism , Ion Channels/physiology , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mice, Inbred BALB C , Microscopy, Confocal , Pain/chemically induced , Pain/metabolism , Pain/prevention & control , Protein Multimerization/immunology , Rats , Receptors, Purinergic P2X2/chemistry , Receptors, Purinergic P2X2/metabolism , Receptors, Purinergic P2X3/chemistry , Receptors, Purinergic P2X3/metabolism , Trinitrobenzenesulfonic Acid , Visceral Pain/chemically induced , Visceral Pain/metabolism , Visceral Pain/prevention & controlABSTRACT
The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR.
Subject(s)
Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Carbamates/chemistry , Citrulline/chemistry , Immunoconjugates/chemistry , Valine/chemistry , Animals , Antibodies, Monoclonal/pharmacokinetics , Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biomarkers , Carboxylesterase/chemistry , Carboxylesterase/metabolism , Drug Design , Drug Stability , Humans , Immunoconjugates/pharmacokinetics , Immunoconjugates/pharmacology , Mice , Mice, Knockout , Models, Molecular , Molecular Conformation , Molecular Structure , Protein Binding , Structure-Activity RelationshipABSTRACT
Applications of microbial transglutaminase (mTGase) produced from Streptomyces mobarensis (S. mobarensis) were recently extended from food to pharmaceutical industry. To use mTGase for clinical applications, like generation of site specific antibody drug conjugates, it would be beneficial to manufacture mTGase in Escherichia coli (E. coli). To date, attempts to express recombinant soluble and active S. mobarensis mTGase have been largely unsuccessful. mTGase from S. mobarensis is naturally expressed as proenzyme and stepwise proteolytically processed into its active mature form outside of the bacterial cell. The pro-domain is essential for correct folding of mTGase as well as for inhibiting activity of mTGase inside the cell. Here, we report a genetically modified mTGase that has full activity and can be expressed at high yields in the cytoplasm of E. coli. To achieve this we performed an alanine-scan of the mTGase pro-domain and identified mutants that maintain its chaperone function but destabilize the cleaved pro-domain/mTGase interaction in a temperature dependent fashion. This allows proper folding of mTGase and keeps the enzyme inactive during expression at 20°C, but results in full activity when shifted to 37°C due to loosen domain interactions. The insertion of the 3C protease cleavage site together with pro-domain alanine mutants Tyr14, Ile24, or Asn25 facilitate high yields (30-75 mg/L), and produced an enzyme with activity identical to wild type mTGase from S. mobarensis. Site-specific antibody drug conjugates made with the E .coli produced mTGase demonstrated identical potency in an in vitro cell assay to those made with mTGase from S. mobarensis.
Subject(s)
Escherichia coli/genetics , Immunoconjugates/metabolism , Protein Engineering , Streptomyces/enzymology , Transglutaminases/genetics , Transglutaminases/metabolism , Amino Acid Sequence , Base Sequence , Enzyme Precursors/chemistry , Enzyme Precursors/genetics , Enzyme Precursors/isolation & purification , Enzyme Precursors/metabolism , Gene Expression , Industrial Microbiology , Models, Molecular , Molecular Sequence Data , Mutagenesis , Mutation , Plasmids/genetics , Protein Structure, Tertiary , Solubility , Streptomyces/chemistry , Streptomyces/genetics , Transglutaminases/chemistry , Transglutaminases/isolation & purificationABSTRACT
The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.
Subject(s)
Aminobenzoates/pharmacokinetics , Drug Carriers/pharmacokinetics , Oligopeptides/pharmacokinetics , Aminobenzoates/administration & dosage , Aminobenzoates/chemistry , Animals , Antibodies/administration & dosage , Drug Carriers/administration & dosage , Drug Carriers/chemistry , Drug Stability , Female , HEK293 Cells , Humans , Macaca fascicularis , Mice, SCID , Oligopeptides/administration & dosage , Oligopeptides/chemistry , RatsSubject(s)
Immunoconjugates , Animals , Cell Line, Tumor , Chemistry, Pharmaceutical , Humans , Hydrophobic and Hydrophilic Interactions , Immunoconjugates/administration & dosage , Immunoconjugates/chemistry , Immunoconjugates/pharmacology , Immunoconjugates/therapeutic use , Mice , Models, Molecular , Rats , Xenograft Model Antitumor AssaysABSTRACT
The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.
Subject(s)
Aminobenzoates/chemistry , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Immunoconjugates/chemistry , Oligopeptides/chemistry , Pancreatic Neoplasms/drug therapy , Aminobenzoates/blood , Aminobenzoates/pharmacokinetics , Aminobenzoates/pharmacology , Animals , Antineoplastic Agents/blood , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Carbamates/chemistry , Cathepsin B/chemistry , Cathepsin B/metabolism , Cell Line, Tumor , Dipeptides/chemistry , Drug Delivery Systems/methods , Drug Stability , Female , Humans , Immunoconjugates/blood , Immunoconjugates/pharmacokinetics , Immunoconjugates/pharmacology , Mice , Mice, Nude , Models, Molecular , Oligopeptides/blood , Oligopeptides/pharmacokinetics , Oligopeptides/pharmacology , Pancreatic Neoplasms/blood , Pancreatic Neoplasms/pathology , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
PURPOSE: To investigate the effectiveness of a high-affinity human monoclonal antibody Fab fragment to Staphylococcus aureus alpha-toxin (LTM14 Fab) as therapy for S. aureus keratitis. METHODS: A single topical drop of the LTM14 Fab antibody to alpha-toxin alone, or in 0.006% benzalkonium chloride (BAK), was applied every 30 min to S. aureus-infected rabbit corneas from 9 to 14 hours post-infection. Erosions and pathology were measured at 15 h post-infection. RESULTS: LTM14 Fab with BAK limited corneal erosions better than LTM14 Fab alone (p = 0.036), and both limited erosions compared to untreated eyes (p ≤ 0.0001). Overall pathology was similar in all groups (p ≥ 0.070), but iritis and chemosis were reduced by treatment (p ≤ 0.036). CONCLUSIONS: The high-affinity human monoclonal Fab fragment antibody (LTM14 Fab) to S. aureus alpha-toxin was effective in reducing corneal damage during S. aureus keratitis.
ABSTRACT
The ability of antibodies to bind an antigen with a high degree of affinity and specificity has led them to become the largest and fastest growing class of therapeutic proteins. Clearly identifying the epitope at which they bind their cognate antigen provides insight into their mechanism of action and helps differentiate antibodies that bind the same antigen. Here, we describe a method to precisely and efficiently map the epitopes of a panel of antibodies in parallel over the course of several weeks. This method relies on the combination of rational library design, quantitative yeast surface display and next-generation DNA sequencing and was demonstrated by mapping the epitopes of several antibodies that neutralize alpha toxin from Staphylococcus aureus. The accuracy of this method was confirmed by comparing the results to the co-crystal structure of one antibody and alpha toxin and was further refined by the inclusion of a lower-affinity variant of the antibody. In addition, this method produced quantitative insight into the epitope residues most critical for the antibody-antigen interaction and enabled the relative affinities of each antibody toward alpha toxin variants to be estimated. This affinity estimate serves as a predictor of neutralizing antibody potency and was used to anticipate the ability of each antibody to effectively bind and neutralize naturally occurring alpha toxin variants secreted by strains of S. aureus, including clinically relevant strains. Ultimately this type information can be used to help select the best clinical candidate among a set of antibodies against a given antigen.
Subject(s)
Antibodies, Monoclonal/immunology , Bacterial Toxins/immunology , Epitopes/analysis , Hemolysin Proteins/immunology , High-Throughput Nucleotide Sequencing , Peptide Library , Saccharomyces cerevisiae/immunology , Staphylococcal Infections/prevention & control , Amino Acid Sequence , Antibodies, Monoclonal/genetics , Bacterial Toxins/genetics , Epitope Mapping/methods , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Flow Cytometry , Hemolysin Proteins/genetics , Humans , Molecular Sequence Data , Protein Conformation , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Staphylococcal Infections/immunology , Staphylococcus aureus/genetics , Staphylococcus aureus/immunologyABSTRACT
Nerve growth factor (NGF) is indispensable during normal embryonic development and critical for the amplification of pain signals in adults. Intervention in NGF signaling holds promise for the alleviation of pain resulting from human diseases such as osteoarthritis, cancer and chronic lower back disorders. We developed a fast, high-fidelity method to convert a hybridoma-derived NGF-targeted mouse antibody into a clinical candidate. This method, termed Library Scanning Mutagenesis (LSM), resulted in the ultra-high affinity antibody tanezumab, a first-in-class anti-hyperalgesic specific for an NGF epitope. Functional and structural comparisons between tanezumab and the mouse 911 precursor antibody using neurotrophin-specific cell survival assays and X-ray crystal structures of both Fab-antigen complexes illustrated high fidelity retention of the NGF epitope. These results suggest the potential for wide applicability of the LSM method for optimization of well-characterized antibodies during humanization.
Subject(s)
Antibodies, Monoclonal, Murine-Derived , Antigen-Antibody Complex , Epitopes , Mutagenesis , Nerve Growth Factor , Adult , Animals , Antibodies, Monoclonal, Humanized/chemistry , Antibodies, Monoclonal, Humanized/genetics , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Murine-Derived/chemistry , Antibodies, Monoclonal, Murine-Derived/genetics , Antibodies, Monoclonal, Murine-Derived/immunology , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/genetics , Antigen-Antibody Complex/immunology , Cells, Cultured , Crystallography, X-Ray , Epitopes/chemistry , Epitopes/genetics , Epitopes/immunology , Female , Humans , Low Back Pain/drug therapy , Low Back Pain/immunology , Mice , Neoplasms/drug therapy , Neoplasms/immunology , Nerve Growth Factor/antagonists & inhibitors , Nerve Growth Factor/chemistry , Nerve Growth Factor/genetics , Nerve Growth Factor/immunology , Osteoarthritis/drug therapy , Osteoarthritis/immunology , Pain Management/methods , Protein Structure, Quaternary , Single-Chain AntibodiesABSTRACT
Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending upon the sample availability, and the number and diversity of the interactions being studied.
Subject(s)
Antibodies, Monoclonal/immunology , Biosensing Techniques , Epitopes/immunology , High-Throughput Screening Assays/methods , Staining and Labeling , Humans , Intercellular Signaling Peptides and Proteins/immunology , Interferometry , Progranulins , Surface Plasmon ResonanceABSTRACT
The emergence and spread of multi-drug-resistant strains of Staphylococcus aureus in hospitals and in the community emphasize the urgency for the development of novel therapeutic interventions. Our approach was to evaluate the potential of harnessing the human immune system to guide the development of novel therapeutics. We explored the role of preexisting antibodies against S. aureus α-hemolysin in the serum of human individuals by isolating and characterizing one antibody with a remarkably high affinity to α-hemolysin. The antibody provided protection in S. aureus pneumonia, skin, and bacteremia mouse models of infection and also showed therapeutic efficacy when dosed up to 18 h post-infection in the pneumonia model. Additionally, in pneumonia and bacteremia animal models, the therapeutic efficacy of the α-hemolysin antibody appeared additive to the antibiotic linezolid. To better understand the mechanism of action of this isolated antibody, we solved the crystal structure of the α-hemolysin:antibody complex. To our knowledge, this is the first report of the crystal structure of the α-hemolysin monomer. The structure of the complex shows that the antibody binds α-hemolysin between the cap and the rim domains. In combination with biochemical data, the structure suggests that the antibody neutralizes the activity of the toxin by preventing binding to the plasma membrane of susceptible host cells. The data presented here suggest that protective antibodies directed against S. aureus molecules exist in some individuals and that such antibodies have a therapeutic potential either alone or in combination with antibiotics.
Subject(s)
Antibodies, Bacterial/administration & dosage , Antibodies, Bacterial/therapeutic use , Antigen-Antibody Complex/administration & dosage , Bacterial Toxins/antagonists & inhibitors , Bacterial Toxins/immunology , Hemolysin Proteins/antagonists & inhibitors , Hemolysin Proteins/immunology , Staphylococcal Infections/immunology , Staphylococcal Infections/prevention & control , Staphylococcus aureus/immunology , Animals , Antibodies, Bacterial/pharmacology , Antibodies, Blocking/blood , Antibodies, Blocking/chemistry , Antibodies, Blocking/therapeutic use , Antigen-Antibody Complex/chemistry , Antigen-Antibody Reactions/immunology , Bacterial Toxins/chemistry , Crystallography, X-Ray , Disease Models, Animal , Female , Hemolysin Proteins/chemistry , Humans , Mice , Mice, Inbred BALB C , Protein Structure, Tertiary , Staphylococcal Infections/blood , Staphylococcus aureus/pathogenicityABSTRACT
Spinal muscular atrophy with respiratory distress type 1 (SMARD1) is a fatal autosomal recessive disorder seen in infants. It is characterized by lower motor neuron degeneration, progressive muscle paralysis and respiratory failure, for which no effective treatment exists. The phenotype of neuromuscular degeneration (nmd) mice closely resembles the human SMARD1. The identification of the mutated mouse gene in nmd mice, Ighmbp2, led to the discovery of mutations of the homologous gene in humans with SMARD1. We have studied the nmd mouse model with in vivo electrophysiological techniques and evaluated the efficacy of Mab2256, a monoclonal antibody with agonist effect on the tyrosine kinase receptor C, trkC, on disease progression in nmd mice. Treatment with Mab2256 resulted in a significant but transient improvement of muscle strength in nmd mice, as well as normalization of the neuromuscular depression during high-frequency nerve stimulation. These results suggest the potential of using monoclonal agonist antibodies for neurotrophin receptors in lower motor neuron diseases such as SMARD1.