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1.
Commun Biol ; 4(1): 1048, 2021 09 08.
Article in English | MEDLINE | ID: mdl-34497355

ABSTRACT

In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.


Subject(s)
Histocompatibility Antigens Class I/metabolism , Immunoglobulin Fc Fragments/metabolism , Receptors, Fc/metabolism , Animals , Half-Life , Histocompatibility Antigens Class I/pharmacology , Humans , Immunoglobulin Fc Fragments/pharmacology , Mice , Mice, Transgenic , Protein Engineering
3.
Sci Transl Med ; 13(590)2021 04 21.
Article in English | MEDLINE | ID: mdl-33883272

ABSTRACT

Cardiovascular disease (CVD) is the leading global cause of death, and treatments that further reduce CV risk remain an unmet medical need. Epidemiological studies have consistently identified low high-density lipoprotein cholesterol (HDL-C) as an independent risk factor for CVD, making HDL elevation a potential clinical target for improved CVD resolution. Endothelial lipase (EL) is a circulating enzyme that regulates HDL turnover by hydrolyzing HDL phospholipids and driving HDL particle clearance. Using MEDI5884, a first-in-class, EL-neutralizing, monoclonal antibody, we tested the hypothesis that pharmacological inhibition of EL would increase HDL-C by enhancing HDL stability. In nonhuman primates, MEDI5884 treatment resulted in lasting, dose-dependent elevations in HDL-C and circulating phospholipids, confirming the mechanism of EL action. We then showed that a favorable lipoprotein profile of elevated HDL-C and reduced low-density lipoprotein cholesterol (LDL-C) could be achieved by combining MEDI5884 with a PCSK9 inhibitor. Last, when tested in healthy human volunteers, MEDI5884 not only raised HDL-C but also increased HDL particle numbers and average HDL size while enhancing HDL functionality, reinforcing EL neutralization as a viable clinical approach aimed at reducing CV risk.


Subject(s)
Lipoproteins, HDL , Proprotein Convertase 9 , Animals , Antibodies, Monoclonal , Cholesterol, HDL , Lipase , Primates
4.
Sci Rep ; 10(1): 17257, 2020 10 14.
Article in English | MEDLINE | ID: mdl-33057063

ABSTRACT

Neuregulin protein 1 (NRG1) is a large (> 60-amino-acid) natural peptide ligand for the ErbB protein family members HER3 and HER4. We developed an agonistic antibody modality, termed antibody ligand mimetics (ALM), by incorporating complex ligand agonists such as NRG1 into an antibody scaffold. We optimized the linker and ligand length to achieve native ligand activity in HEK293 cells and cardiomyocytes derived from induced pluripotent stem cells (iPSCs) and used a monomeric Fc-ligand fusion platform to steer the ligand specificity toward HER4-dominant agonism. With the help of selectivity engineering, these enhanced ALM molecules can provide an antibody scaffold with increased receptor specificity and the potential to greatly improve the pharmacokinetics, stability, and downstream developability profiles from the natural ligand approach. This ligand mimetic design and optimization approach can be expanded to apply to other cardiovascular disease targets and emerging therapeutic areas, providing differentiated drug molecules with increased specificity and extended half-life.


Subject(s)
Antibodies, Monoclonal/chemistry , Neuregulin-1/chemistry , Receptor, ErbB-4/agonists , Antibodies, Monoclonal/metabolism , HEK293 Cells , Humans , Induced Pluripotent Stem Cells/chemistry , Induced Pluripotent Stem Cells/metabolism , Kinetics , Ligands , Myocytes, Cardiac/chemistry , Myocytes, Cardiac/metabolism , Neuregulin-1/metabolism , Protein Binding , Receptor, ErbB-4/metabolism , Signal Transduction
5.
PLoS One ; 15(6): e0234268, 2020.
Article in English | MEDLINE | ID: mdl-32497150

ABSTRACT

Annexin A1 (anxA1) is an immunomodulatory protein that has been proposed as a tumor vascular target for antitumor biologic agents, yet to date the vascular expression of anxA1 in specific tumor indications has not been systematically assessed. Attempts to evaluate vascular anxA1 expression by immunohistochemistry are complicated by a lack of available antibodies that are both specific for anxA1 and bind the N-terminal-truncated form of anxA1 that has previously been identified in tumor vasculature. To study the vascular expression pattern of anxA1 in non-small-cell lung carcinoma (NSCLC), we isolated an antibody capable of binding N-terminal-truncated anxA127-346 and employed it in immunohistochemical studies of human lung specimens. Lung tumor specimens evaluated with this antibody revealed vascular (endothelial) anxA1 expression in five of eight tumor samples studied, but no vascular anxA1 expression was observed in normal lung tissue. Tumor microarray analysis further demonstrated positive vascular staining for anxA1 in 30 of 80 NSCLC samples, and positive staining of neoplastic cells was observed in 54 of 80 samples. No correlation was observed between vascular and parenchymal anxA1 expression. Two rodent tumor models, B16-F10 and Py230, were determined to have upregulated anxA1 expression in the intratumoral vasculature. These data validate anxA1 as a potential vascular anti-tumor target in a subset of human lung tumors and identify rodent models which demonstrate anxA1 expression in tumor vasculature.


Subject(s)
Annexin A1/metabolism , Carcinoma, Non-Small-Cell Lung/metabolism , Lung Neoplasms/metabolism , Up-Regulation , Animals , Carcinoma, Non-Small-Cell Lung/blood supply , Cell Line, Tumor , Disease Models, Animal , Female , Humans , Lung Neoplasms/blood supply , Mice
6.
PLoS Comput Biol ; 15(5): e1006980, 2019 05.
Article in English | MEDLINE | ID: mdl-31042706

ABSTRACT

Antibodies are an important class of therapeutics that have significant clinical impact for the treatment of severe diseases. Computational tools to support antibody drug discovery have been developing at an increasing rate over the last decade and typically rely upon a predetermined co-crystal structure of the antibody bound to the antigen for structural predictions. Here, we show an example of successful in silico affinity maturation of a hybridoma derived antibody, AB1, using just a homology model of the antibody fragment variable region and a protein-protein docking model of the AB1 antibody bound to the antigen, murine CCL20 (muCCL20). In silico affinity maturation, together with alanine scanning, has allowed us to fine-tune the protein-protein docking model to subsequently enable the identification of two single-point mutations that increase the affinity of AB1 for muCCL20. To our knowledge, this is one of the first examples of the use of homology modelling and protein docking for affinity maturation and represents an approach that can be widely deployed.


Subject(s)
Antibody Affinity/physiology , Computational Biology/methods , Amino Acid Sequence , Animals , Antibodies/chemistry , Chemokine CCL20 , Computer Simulation , Drug Design , Immunoglobulin Variable Region , Mice , Models, Molecular , Protein Binding , Protein Conformation
7.
Mol Pharm ; 15(12): 5697-5710, 2018 12 03.
Article in English | MEDLINE | ID: mdl-30395473

ABSTRACT

Monoclonal antibodies (mAbs) are complex molecular structures. They are often prone to development challenges particularly at high concentrations due to undesired solution properties such as reversible self-association, high viscosity, and liquid-liquid phase separation. In addition to formulation optimization, applying protein engineering can provide an alternative mitigation strategy. Protein engineering during the discovery phase can provide great benefits to optimize molecular properties, resulting in improved developability profiles. Here, we present a case study utilizing complementary analytical and predictive in silico methods. We have systematically identified and reengineered problematic residues responsible for the self-association of a model mAb, driven by a complex combination of hydrophobic and electrostatic interactions. Noteworthy findings include a more dominant contribution of hydrophobic interactions to self-association and potential feasibility of mutations in the CDR regions to mitigate self-association. The engineered mutation panel enabled us to assess potential correlations among commonly utilized developability screening assays, including affinity capture self-interaction nanospectroscopy (AC-SINS), dynamic light scattering (DLS), and apparent solubility by PEG-precipitation. In addition, we evaluated the correlations between experimental measurements and computational predictions. CamSol, an in silico computational tool that accounts for complex molecular interactions and neighboring hotspots, was found to be an effective screening tool. Our work led to reengineered mAb variants, better suited for high-concentration liquid formulation development. The engineered mAbs exhibited enhanced in vitro and simulated in vivo solubility and reduced self-association propensity, while maintaining binding affinity and thermal stability.


Subject(s)
Antibodies, Monoclonal/genetics , Drug Development , Mutagenesis, Site-Directed , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/pharmacokinetics , Biological Availability , Chemistry, Pharmaceutical , Cloning, Molecular , Computer Simulation , Drug Stability , Hydrophobic and Hydrophilic Interactions , Models, Biological , Models, Chemical , Mutation , Solubility , Static Electricity , Viscosity
8.
PLoS One ; 11(8): e0160345, 2016.
Article in English | MEDLINE | ID: mdl-27479095

ABSTRACT

The immunoglobulin Fc region is a homodimer consisted of two sets of CH2 and CH3 domains and has been exploited to generate two-arm protein fusions with high expression yields, simplified purification processes and extended serum half-life. However, attempts to generate one-arm fusion proteins with monomeric Fc, with one set of CH2 and CH3 domains, are often plagued with challenges such as weakened binding to FcRn or partial monomer formation. Here, we demonstrate the generation of a stable IgG4 Fc monomer with a unique combination of mutations at the CH3-CH3 interface using rational design combined with in vitro evolution methodologies. In addition to size-exclusion chromatography and analytical ultracentrifugation, we used multi-angle light scattering (MALS) to show that the engineered Fc monomer exhibits excellent monodispersity. Furthermore, crystal structure analysis (PDB ID: 5HVW) reveals monomeric properties supported by disrupted interactions at the CH3-CH3 interface. Monomeric Fc fusions with Fab or scFv achieved FcRn binding and serum half-life comparable to wildtype IgG. These results demonstrate that this monomeric IgG4 Fc is a promising therapeutic platform to extend the serum half-life of proteins in a monovalent format.


Subject(s)
Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Animals , Chromatography, Gel , Crystallography, X-Ray , Dynamic Light Scattering , Enzyme-Linked Immunosorbent Assay , Half-Life , Humans , Immunoglobulin Fc Fragments/blood , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/genetics , Immunoglobulin G/blood , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Mice , Mice, Transgenic , Molecular Dynamics Simulation , Peptide Library , Protein Binding , Protein Domains , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Ultracentrifugation
9.
MAbs ; 8(3): 454-67, 2016.
Article in English | MEDLINE | ID: mdl-26854859

ABSTRACT

CD73 (ecto-5'-nucleotidase) has recently been established as a promising immuno-oncology target. Given its role in activating purinergic signaling pathways to elicit immune suppression, antagonizing CD73 (i.e., releasing the brake) offers a complimentary pathway to inducing anti-tumor immune responses. Here, we describe the mechanistic activity of a new clinical therapeutic, MEDI9447, a human monoclonal antibody that non-competitively inhibits CD73 activity. Epitope mapping, structural, and mechanistic studies revealed that MEDI9447 antagonizes CD73 through dual mechanisms of inter-CD73 dimer crosslinking and/or steric blocking that prevent CD73 from adopting a catalytically active conformation. To our knowledge, this is the first report of an antibody that inhibits an enzyme's function through 2 distinct modes of action. These results provide a finely mapped epitope that can be targeted for selective, potent, and non-competitive inhibition of CD73, as well as establish a strategy for inhibiting enzymes that function in both membrane-bound and soluble states.


Subject(s)
5'-Nucleotidase , Adenosine Monophosphate , Antibodies, Monoclonal/chemistry , Antineoplastic Agents/chemistry , Enzyme Inhibitors/chemistry , 5'-Nucleotidase/antagonists & inhibitors , 5'-Nucleotidase/chemistry , Adenosine Monophosphate/chemistry , GPI-Linked Proteins/antagonists & inhibitors , GPI-Linked Proteins/chemistry , Humans , Hydrolysis
10.
MAbs ; 8(1): 163-75, 2016.
Article in English | MEDLINE | ID: mdl-26514996

ABSTRACT

Antagonistic antibodies targeting the G-protein C-X-C chemokine receptor 4 (CXCR4) hold promising therapeutic potential in various diseases. We report for the first time the detailed mechanism of action at a molecular level of a potent anti-CXCR4 antagonistic antibody (MEDI3185). We characterized the MEDI3185 paratope using alanine scanning on all 6 complementary-determining regions (CDRs). We also mapped its epitope using CXCR4 mutagenesis to assess the relative importance of the CXCR4 N-terminal peptide, extracellular loops (ECL) and ligand-binding pocket. We show that the interaction between MEDI3185 and CXCR4 is mediated mostly by CDR3H in MEDI3185 and ECL2 in CXCR4. The MEDI3185 epitope comprises the entire ECL2 sequence, lacks any so-called 'hot-spot' and is remarkably resistant to mutations. The structure of MEDI3185 variable domains was modeled, and suggested a ß-strand/ß-strand interaction between MEDI3185 CDR3H and CXCR4 ECL2, resulting in direct steric hindrance with CXCR4 ligand SDF-1. These findings may have important implications for designing antibody therapies against CXCR4.


Subject(s)
Antibodies, Monoclonal/immunology , Receptors, CXCR4/antagonists & inhibitors , Receptors, CXCR4/immunology , Animals , CHO Cells , Cricetinae , Cricetulus , Humans , MCF-7 Cells , Mice , Peptides/antagonists & inhibitors , Peptides/immunology , Protein Structure, Secondary , Protein Structure, Tertiary
11.
MAbs ; 7(2): 428-39, 2015.
Article in English | MEDLINE | ID: mdl-25606664

ABSTRACT

Anifrolumab (anifrolumab) is an antagonist human monoclonal antibody that targets interferon α receptor 1 (IFNAR1). Anifrolumab has been developed to treat autoimmune diseases and is currently in clinical trials. To decipher the molecular basis of its mechanism of action, we engaged in multiple epitope mapping approaches to determine how it interacts with IFNAR1 and antagonizes the receptor. We identified the epitope of anifrolumab using enzymatic fragmentation, phage-peptide library panning and mutagenesis approaches. Our studies revealed that anifrolumab recognizes the SD3 subdomain of IFNAR1 with the critical residue R(279). Further, we solved the crystal structure of anifrolumab Fab to a resolution of 2.3 Å. Guided by our epitope mapping studies, we then used in silico protein docking of the anifrolumab Fab crystal structure to IFNAR1 and characterized the corresponding mode of binding. We find that anifrolumab sterically inhibits the binding of IFN ligands to IFNAR1, thus blocking the formation of the ternary IFN/IFNAR1/IFNAR2 signaling complex. This report provides the molecular basis for the mechanism of action of anifrolumab and may provide insights toward designing antibody therapies against IFNAR1.


Subject(s)
Antibodies, Monoclonal/chemistry , Epitope Mapping , Epitopes/chemistry , Peptide Library , Receptor, Interferon alpha-beta/antagonists & inhibitors , Receptor, Interferon alpha-beta/chemistry , Amino Acid Substitution , Animals , Antibodies, Monoclonal/therapeutic use , Autoimmune Diseases/drug therapy , Autoimmune Diseases/metabolism , CHO Cells , Cricetinae , Cricetulus , Epitopes/genetics , Interferons/antagonists & inhibitors , Interferons/chemistry , Interferons/deficiency , Interferons/metabolism , Male , Mutation, Missense , Receptor, Interferon alpha-beta/genetics , Receptor, Interferon alpha-beta/metabolism
12.
PLoS One ; 9(11): e112070, 2014.
Article in English | MEDLINE | ID: mdl-25372291

ABSTRACT

Surface plasmon resonance (SPR)-based immunoassays have numerous applications and require high affinity reagents for sensitive and reliable measurements. We describe a quick approach to turn low affinity antibodies into appropriate capture reagents. We used antibodies recognizing human ephrin type A receptor 2 (EphA2) and a ProteOn XPR36 as a model system. We generated so-called 'bi-epitope' sensor surfaces by immobilizing various pairs of anti-EphA2 antibodies using standard amine coupling. The apparent binding affinities to EphA2 and EphA2 detection sensitivities of the bi-epitope and 'single-epitope' surfaces were then compared. For all antibody pairs tested, bi-epitope surfaces exhibited an ∼ 10-100-fold improvement in apparent binding affinities when compared with single-epitope ones. When pairing 2 antibodies of low intrinsic binding affinities (∼ 10(-8) M) and fast dissociation rates (∼ 10(-2) s(-1)), the apparent binding affinity and dissociation rate of the bi-epitope surface was improved up to ∼ 10(-10) M and 10(-4) s(-1), respectively. This led to an ∼ 100-200-fold enhancement in EphA2 limit of detection in crude cell supernatants. Our results show that the use of antibody mixtures in SPR applications constitutes a powerful approach to develop sensitive immunoassays, as previously shown for non-SPR formats. As SPR-based assays have significantly expanded their reach in the last decade, such an approach promises to further accelerate their development.


Subject(s)
Antibodies, Monoclonal, Murine-Derived/chemistry , Receptor, EphA2/chemistry , Surface Plasmon Resonance , Antibody Affinity , Epitopes , Humans
13.
J Biol Chem ; 289(43): 29874-80, 2014 Oct 24.
Article in English | MEDLINE | ID: mdl-25210036

ABSTRACT

MEDI4893 is a neutralizing human monoclonal antibody that targets α-toxin (AT) and is currently undergoing evaluation in the field of Staphylococcus aureus-mediated diseases. We have solved the crystal structure of MEDI4893 Fab bound to monomeric AT at a resolution of 2.56 Å and further characterized its epitope using various engineered AT variants. We have found that MEDI4893 recognizes a novel epitope in the so-called "rim" domain of AT and exerts its neutralizing effect through a dual mechanism. In particular, MEDI4893 not only sterically blocks binding of AT to its cell receptor but also prevents it from adopting a lytic heptameric trans-membrane conformation.


Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Neutralizing/immunology , Bacterial Toxins/immunology , Hemolysin Proteins/immunology , Neutralization Tests , Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal, Humanized , Antibodies, Neutralizing/chemistry , Broadly Neutralizing Antibodies , Cell Line , Crystallography, X-Ray , Epitope Mapping , Humans , Immunoglobulin Fab Fragments/chemistry , Models, Molecular , Protein Binding , Recombinant Proteins/chemistry , Surface Plasmon Resonance
14.
Immunology ; 142(4): 526-35, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24635566

ABSTRACT

A number of different methods are commonly used to map the fine details of the interaction between an antigen and an antibody. Undoubtedly the method that is now most commonly used to give details at the level of individual amino acids and atoms is X-ray crystallography. The feasibility of undertaking crystallographic studies has increased over recent years through the introduction of automation, miniaturization and high throughput processes. However, this still requires a high level of sophistication and expense and cannot be used when the antigen is not amenable to crystallization. Nuclear magnetic resonance spectroscopy offers a similar level of detail to crystallography but the technical hurdles are even higher such that it is rarely used in this context. Mutagenesis of either antigen or antibody offers the potential to give information at the amino acid level but suffers from the uncertainty of not knowing whether an effect is direct or indirect due to an effect on the folding of a protein. Other methods such as hydrogen deuterium exchange coupled to mass spectrometry and the use of short peptides coupled with ELISA-based approaches tend to give mapping information over a peptide region rather than at the level of individual amino acids. It is quite common to use more than one method because of the limitations and even with a crystal structure it can be useful to use mutagenesis to tease apart the contribution of individual amino acids to binding affinity.


Subject(s)
Antigen-Antibody Complex/chemistry , Deuterium Exchange Measurement/methods , Mutagenesis , Nuclear Magnetic Resonance, Biomolecular/methods , Animals , Antigen-Antibody Complex/genetics , Crystallography, X-Ray , Humans
15.
J Biol Chem ; 289(11): 7812-24, 2014 Mar 14.
Article in English | MEDLINE | ID: mdl-24469444

ABSTRACT

We report the three-dimensional structure of human neonatal Fc receptor (FcRn) bound concurrently to its two known ligands. More particularly, we solved the crystal structure of the complex between human FcRn, wild-type human serum albumin (HSA), and a human Fc engineered for improved pharmacokinetics properties (Fc-YTE). The crystal structure of human FcRn bound to wild-type HSA alone is also presented. HSA domain III exhibits an extensive interface of contact with FcRn, whereas domain I plays a lesser role. A molecular explanation for the HSA recycling mechanism is provided with the identification of FcRn His(161) as the only potential direct contributor to the corresponding pH-dependent process. At last, this study also allows an accurate structural definition of residues considered for decades as important to the human IgG/FcRn interaction and reveals Fc His(310) as a significant contributor to pH-dependent binding. Finally, we explain various structural mechanisms by which several Fc mutations (including YTE) result in increased human IgG binding to FcRn. Our study provides an unprecedented relevant understanding of the molecular basis of human Fc interaction with human FcRn.


Subject(s)
Histocompatibility Antigens Class I/chemistry , Immunoglobulin G/chemistry , Receptors, Fc/chemistry , Serum Albumin/chemistry , Crystallization , Crystallography, X-Ray , HEK293 Cells , Humans , Hydrogen Bonding , Hydrogen-Ion Concentration , Immunoglobulin Fc Fragments/chemistry , Ligands , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Recombinant Proteins/chemistry
16.
J Mol Biol ; 413(2): 390-405, 2011 Oct 21.
Article in English | MEDLINE | ID: mdl-21867711

ABSTRACT

We report here the three-dimensional structure of human ephrin type A receptor 2 (EphA2) bound to the Fab (fragment antigen binding) of an agonistic human antibody (1C1; IgG1/κ). The structure of the corresponding complex was solved at a resolution of 2.5 Å using molecular replacement and constitutes the first reported structure of a human ephrin receptor bound to an antibody. We have also defined the corresponding functional epitope using a mutagenesis-based approach. This study revealed discrete structural features that determine the fine specificity of 1C1 to EphA2. Our data also provided a molecular basis for 1C1 mechanism of action. More precisely, we propose that its agonistic, internalizing properties are the result of ligand mimicry by the third heavy-chain complementarity-determining region of 1C1. Because EphA2 is an important contributor to cancer formation and progression, these findings may have implications for designing the next generation of anti-tumor therapies.


Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Antibodies, Monoclonal/chemistry , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin G/chemistry , Immunoglobulin kappa-Chains/chemistry , Receptor, EphA2/agonists , Amino Acid Sequence , Animals , Antibodies, Anti-Idiotypic/immunology , Antibodies, Anti-Idiotypic/metabolism , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/metabolism , Crystallography, X-Ray , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin kappa-Chains/immunology , Immunoglobulin kappa-Chains/metabolism , Male , Mice , Models, Chemical , Molecular Sequence Data , Phosphorylation , Prostatic Neoplasms/metabolism , Protein Structure, Secondary , Receptor, EphA2/immunology , Receptor, EphA2/metabolism , Sequence Homology, Amino Acid , Tumor Cells, Cultured
17.
Article in English | MEDLINE | ID: mdl-20516612

ABSTRACT

The recombinant N-terminal domain of human ephrin type-A receptor 2 (rEphA2) has been crystallized in complex with the recombinantly produced Fab fragment of a fully human antibody (1C1; IgG1/kappa). These are the first reported crystals of an ephrin receptor bound to an antibody. The orthorhombic crystals belonged to space group C222(1) (the 00l reflections obey the l = 2n rule), with unit-cell parameters a = 78.93, b = 120.79, c = 286.20 A. The diffraction of the crystals extended to 2.0 A resolution. However, only data to 2.55 A resolution were considered to be useful owing to spot overlap caused by the long unit-cell parameter. The asymmetric unit is most likely to contain two 1C1 Fab-rEphA2 complexes. This corresponds to a crystal volume per protein weight (V(M)) of 2.4 A(3) Da(-1) and a solvent content of 49.5%. The three-dimensional structure of this complex will shed light on the molecular basis of 1C1 specificity. This will also contribute to a better understanding of the mechanism of action of this antibody, the current evaluation of which as an antibody-drug conjugate in cancer therapy makes it a particularly interesting case study.


Subject(s)
Antigen-Antibody Complex/chemistry , Immunoglobulin Fab Fragments/chemistry , Peptide Fragments/chemistry , Receptor, EphA2/chemistry , Antigen-Antibody Complex/immunology , Crystallization , Crystallography, X-Ray , Humans , Immunoglobulin Fab Fragments/immunology , Peptide Fragments/immunology , Receptor, EphA2/immunology
18.
J Allergy Clin Immunol ; 125(6): 1344-1353.e2, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20513525

ABSTRACT

BACKGROUND: Peripheral blood eosinophilia and lung mucosal eosinophil infiltration are hallmarks of bronchial asthma. IL-5 is a critical cytokine for eosinophil maturation, survival, and mobilization. Attempts to target eosinophils for the treatment of asthma by means of IL-5 neutralization have only resulted in partial removal of airway eosinophils, and this warrants the development of more effective interventions to further explore the role of eosinophils in the clinical expression of asthma. OBJECTIVE: We sought to develop a novel humanized anti-IL-5 receptor alpha (IL-5Ralpha) mAb with enhanced effector function (MEDI-563) that potently depletes circulating and tissue-resident eosinophils and basophils for the treatment of asthma. METHODS: We used surface plasmon resonance to determine the binding affinity of MEDI-563 to FcgammaRIIIa. Primary human eosinophils and basophils were used to demonstrate antibody-dependent cell-mediated cytotoxicity. The binding epitope of MEDI-563 on IL-5Ralpha was determined by using site-directed mutagenesis. The consequences of MEDI-563 administration on peripheral blood and bone marrow eosinophil depletion was investigated in nonhuman primates. RESULTS: MEDI-563 binds to an epitope on IL-5Ralpha that is in close proximity to the IL-5 binding site, and it inhibits IL-5-mediated cell proliferation. MEDI-563 potently induces antibody-dependent cell-mediated cytotoxicity of both eosinophils (half-maximal effective concentration = 0.9 pmol/L) and basophils (half-maximal effective concentration = 0.5 pmol/L) in vitro. In nonhuman primates MEDI-563 depletes blood eosinophils and eosinophil precursors in the bone marrow. CONCLUSIONS: MEDI-563 might provide a novel approach for the treatment of asthma through active antibody-dependent cell-mediated depletion of eosinophils and basophils rather than through passive removal of IL-5.


Subject(s)
Antibodies, Monoclonal/administration & dosage , Eosinophils/metabolism , Epitopes/metabolism , Interleukin-5 Receptor alpha Subunit/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Antibodies, Monoclonal/adverse effects , Antibody Affinity , Antibody-Dependent Cell Cytotoxicity , Cell Count , Eosinophils/drug effects , Eosinophils/pathology , Epitope Mapping , Female , Humans , Interleukin-5 Receptor alpha Subunit/genetics , Interleukin-5 Receptor alpha Subunit/immunology , Macaca fascicularis , Male , Mutagenesis, Site-Directed , Protein Engineering , Receptors, IgG/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Surface Plasmon Resonance
19.
Mol Immunol ; 46(8-9): 1750-5, 2009 May.
Article in English | MEDLINE | ID: mdl-19250681

ABSTRACT

The first three-dimensional structure of a human Fc fragment genetically engineered for improved pharmacokinetics properties is reported. When introduced into the C(H)2 domain of human immunoglobulin G (IgG) molecules, the triple mutation M252Y/S254T/T256E ('YTE') causes an about 10-fold increase in their binding to the human neonatal Fc receptor (FcRn). This translates into an almost 4-fold increase in the serum half-life of YTE-containing human IgGs in cynomolgus monkeys. A recombinantly produced human Fc/YTE fragment was crystallized and its structure solved at a resolution of 2.5A using molecular replacement. This revealed that Fc/YTE three-dimensional structure is very similar to that of other human Fc fragments in the experimentally visible region spanning residues 236-444. We propose that the enhanced interaction between Fc/YTE and human FcRn is likely mediated by local effects at the substitutions sites. Molecular modeling suggested that potential favorable hydrogen bonds along with an increase in the surface of contact between the two partners may account in part for the corresponding increase in affinity.


Subject(s)
Immunoglobulin Fc Fragments/blood , Immunoglobulin Fc Fragments/genetics , Protein Engineering/methods , Animals , Cells, Cultured , Crystallography, X-Ray , Glutamic Acid/chemistry , Glutamic Acid/genetics , Half-Life , Histocompatibility Antigens Class I/metabolism , Humans , Immunoglobulin Fc Fragments/chemistry , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/chemistry , Immunoglobulin G/genetics , Models, Molecular , Protein Conformation , Protein Stability , Rats , Receptors, Fc/metabolism , Threonine/chemistry , Threonine/genetics , Tyrosine/chemistry , Tyrosine/genetics
20.
Article in English | MEDLINE | ID: mdl-19153447

ABSTRACT

Recombinant human interferon alpha-2A (rhIFN-alpha-2A) has been crystallized in complex with the recombinantly produced Fab fragment of a therapeutic monoclonal antibody (MEDI545; IgG1/kappa) which targets several human interferon alpha subtypes. This constitutes the first reported crystals of a human type I interferon bound to an antibody. The orthorhombic crystals belonged to either space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 134.82, b = 153.26, c = 163.49 A. The diffraction of the crystals extended to 3.0 A resolution. The asymmetric unit contained two Fab-rhIFN-alpha-2A complexes. This corresponded to a crystal volume per protein weight (V(M)) of 3.02 A(3) Da(-1) and a solvent content of 59.3%. The corresponding three-dimensional structure is expected to shed light on the mechanism of action of MEDI545 and the molecular basis of its specificity.


Subject(s)
Antibodies, Anti-Idiotypic/chemistry , Interferon-alpha/chemistry , Interferons/chemistry , Antibodies, Monoclonal/chemistry , Crystallization , Electrophoresis, Polyacrylamide Gel , Humans , Imaging, Three-Dimensional , Immunoglobulin Fab Fragments/chemistry , Interferon alpha-2 , Molecular Conformation , Molecular Structure , Recombinant Proteins , X-Ray Diffraction
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