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1.
Commun Biol ; 6(1): 997, 2023 09 29.
Article in English | MEDLINE | ID: mdl-37773269

ABSTRACT

Antibody engineering technology is at the forefront of therapeutic antibody development. The primary goal for engineering a therapeutic antibody is the generation of an antibody with a desired specificity, affinity, function, and developability profile. Mature antibodies are considered antigen specific, which may preclude their use as a starting point for antibody engineering. Here, we explore the plasticity of mature antibodies by engineering novel specificity and function to a pre-selected antibody template. Using a small, focused library, we engineered AAL160, an anti-IL-1ß antibody, to bind the unrelated antigen IL-17A, with the introduction of seven mutations. The final redesigned antibody, 11.003, retains favorable biophysical properties, binds IL-17A with sub-nanomolar affinity, inhibits IL-17A binding to its cognate receptor and is functional in a cell-based assay. The epitope of the engineered antibody can be computationally predicted based on the sequence of the template antibody, as is confirmed by the crystal structure of the 11.003/IL-17A complex. The structures of the 11.003/IL-17A and the AAL160/IL-1ß complexes highlight the contribution of germline residues to the paratopes of both the template and re-designed antibody. This case study suggests that the inherent plasticity of antibodies allows for re-engineering of mature antibodies to new targets, while maintaining desirable developability profiles.


Subject(s)
Antibodies , Interleukin-17 , Epitopes/chemistry , Antigens , Binding Sites, Antibody
2.
Proteins ; 87(3): 185-197, 2019 03.
Article in English | MEDLINE | ID: mdl-30520519

ABSTRACT

We combined computational and experimental methods to interrogate the binding determinants of angiopoietin-2 (Ang2) to its receptor tyrosine kinase (RTK) Tie2-a central signaling system in angiogenesis, inflammation, and tumorigenesis. We used physics-based electrostatic and surface-area calculations to identify the subset of interfacial Ang2 and Tie2 residues that can affect binding directly. Using random and site-directed mutagenesis and yeast surface display (YSD), we validated these predictions and identified additional Ang2 positions that affected receptor binding. We then used burial-based calculations to classify the larger set of Ang2 residues that are buried in the Ang2 core, whose mutations can perturb the Ang2 structure and thereby affect interactions with Tie2 indirectly. Our analysis showed that the Ang2-Tie2 interface is dominated by nonpolar contributions, with only three Ang2 and two Tie2 residues that contribute electrostatically to intermolecular interactions. Individual interfacial residues contributed only moderately to binding, suggesting that engineering of this interface will require multiple mutations to reach major effects. Conversely, substitutions in substantially buried Ang2 residues were more prevalent in our experimental screen, reduced binding substantially, and are therefore more likely to have a deleterious effect that might contribute to oncogenesis. Computational analysis of additional RTK-ligand complexes, c-Kit-SCF and M-CSF-c-FMS, and comparison to previous YSD results, further show the utility of our combined methodology.


Subject(s)
Multiprotein Complexes/chemistry , Protein Interaction Maps/genetics , Receptor, TIE-2/chemistry , Vesicular Transport Proteins/chemistry , Carcinogenesis/genetics , Computer Simulation , Humans , Inflammation/genetics , Ligands , Multiprotein Complexes/genetics , Mutagenesis, Site-Directed , Mutation/genetics , Neovascularization, Pathologic/genetics , Protein Binding/genetics , Proto-Oncogene Proteins c-kit/chemistry , Receptor, TIE-2/genetics , Signal Transduction/genetics , Stem Cell Factor/chemistry , Vesicular Transport Proteins/genetics
3.
BMC Biol ; 16(1): 92, 2018 08 17.
Article in English | MEDLINE | ID: mdl-30119679

ABSTRACT

BACKGROUND: Increased activity of the receptor tyrosine kinase Tie2 has been implicated in the promotion of pathological angiogenesis. This activity is mainly mediated through angiopoietin (Ang)1- and Ang2-dependent activation of integrins by Tie2, rendering the Ang/Tie2/integrin axis an attractive putative target for cancer therapeutics. RESULTS: To target this axis, we developed single domain, non-immunoglobulin high-affinity bi-specific protein inhibitors against both Tie2 and αvß3 integrin. We have previously engineered the Ang2-binding domain of Tie2 (Ang2-BD) as a Tie2 inhibitor. Here, we engineered an exposed loop in Ang2-BD to generate variants that include an integrin-binding Arg-Gly-Asp (RGD) motif and used flow cytometry screening of a yeast-displayed Ang2-BD RGD loop library to identify the integrin antagonists. The bi-specific antagonists targeting both Tie2 and αvß3 integrin inhibited adhesion and proliferation of endothelial cells cultured together with the αvß3 integrin ligand vitronectin, as well as endothelial cell invasion and tube formation. The bi-specific reagents inhibited downstream signaling by Tie2 intracellularly in response to its agonist Ang1 more effectively than the wild-type Ang2 BD that binds Tie2 alone. CONCLUSIONS: Collectively, this study-the first to describe inhibitors targeting all the known functions resulting from Tie2/integrin αvß3 cross-talk-has created new tools for studying Tie2- and integrin αvß3-dependent molecular pathways and provides the basis for the rational and combinatorial engineering of ligand-Tie2 and ligand-integrin αvß3 receptor interactions. Given the roles of these pathways in cancer angiogenesis and metastasis, this proof of principle study paves the route to create novel Tie2/integrin αvß3-targeting proteins for clinical use as imaging and therapeutic agents.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Neovascularization, Physiologic/genetics , Receptor, TIE-2/antagonists & inhibitors , Receptors, Vitronectin/genetics , Ribonuclease, Pancreatic/antagonists & inhibitors , Angiogenesis Inhibitors/chemistry , Animals , Mice , Receptor, TIE-2/chemistry , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Receptors, Vitronectin/chemistry , Receptors, Vitronectin/metabolism , Ribonuclease, Pancreatic/chemistry , Ribonuclease, Pancreatic/genetics , Ribonuclease, Pancreatic/metabolism
4.
Oncotarget ; 8(20): 33571-33585, 2017 May 16.
Article in English | MEDLINE | ID: mdl-28422724

ABSTRACT

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.


Subject(s)
Angiogenesis Inhibitors/pharmacology , Angiopoietin-2/metabolism , Neovascularization, Pathologic/metabolism , Receptor, TIE-2/antagonists & inhibitors , Angiogenesis Inhibitors/chemistry , Angiopoietin-2/chemistry , Cell Movement/drug effects , Cells, Cultured , Drug Discovery , Drug Screening Assays, Antitumor , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Humans , Ligands , Models, Molecular , Molecular Conformation , Neovascularization, Pathologic/drug therapy , Neovascularization, Physiologic/drug effects , Phosphorylation , Protein Binding , Protein Interaction Domains and Motifs , Receptor, TIE-2/chemistry , Receptor, TIE-2/genetics , Receptor, TIE-2/metabolism , Signal Transduction/drug effects , Small Molecule Libraries , Structure-Activity Relationship
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