ABSTRACT
UNLABELLED: The HIV-1 virion infectivity factor (Vif) targets the cellular cytidine deaminases APOBEC3G (A3G) and APOBEC3F (A3F) for degradation via the host ubiquitin-proteasome pathway. Vif recruits a cellular E3 ubiquitin ligase to polyubiquitinate A3G/F. The activity of Vif critically depends on the cellular core binding factor beta (CBFß). In this study, we investigated the Vif-CBFß interaction and the role of CBFß in the E3 ligase assembly. Vif-CBFß interaction requires an extensive region of Vif spanning most of its amino terminus and zinc finger region, and cullin 5 (Cul5) binding enhances the stability of the Vif-CBFß interaction. Our results further demonstrate that CBFß plays a critical role in facilitating Cul5 binding to the Vif/elongin B/elongin C complex. Vif, with or without bound substrate, is unable to bind Cul5 in the absence of CBFß. These studies support the notion that CBFß serves as a molecular chaperone to facilitate Vif-E3 ligase assembly. IMPORTANCE: The host antiviral restriction factors A3G/F inhibit viral replication. The HIV-1 protein Vif targets A3G/F for degradation. This immune evasion activity of Vif is dependent on the cellular factor CBFß. Multiple regions of Vif are known to be important for Vif function, but the mechanisms are unclear. The studies described here provide important information about the Vif-CBFß interaction interface and the function of CBFß in E3 ligase assembly. In particular, our comprehensive Vif-CBFß interface mapping results help to delineate the role of various Vif regions, determining if they are important for binding CBFß or A3G/F. Furthermore, our studies reveal an important potential mechanism of CBFß that has not been shown before. Our results suggest that CBFß may serve as a molecular chaperone to enable Vif to adopt an appropriate conformation for interaction with the Cul5-based E3 ligase. This study advances our understanding of how CBFß facilitates the Vif-mediated degradation of APOBEC3 proteins.
Subject(s)
Core Binding Factor beta Subunit/metabolism , Cullin Proteins/metabolism , HIV Infections/metabolism , HIV-1/metabolism , Ubiquitin-Protein Ligases/metabolism , vif Gene Products, Human Immunodeficiency Virus/metabolism , Cell Line , Core Binding Factor beta Subunit/genetics , Cullin Proteins/genetics , Elongin , HIV Infections/enzymology , HIV Infections/genetics , HIV Infections/virology , HIV-1/genetics , Humans , Protein Binding , Transcription Factors/genetics , Transcription Factors/metabolism , Ubiquitin-Protein Ligases/genetics , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
The small molecule thioflavin T (ThT) is a defining probe for the identification and mechanistic study of amyloid fiber formation. As such, ThT is fundamental to investigations of serious diseases such as Alzheimer's disease, Parkinson disease, and type II diabetes. For each disease, a different protein undergoes conformational conversion to a ß-sheet rich fiber. The fluorescence of ThT exhibits an increase in quantum yield upon binding these fibers. Despite its widespread use, the structural basis for binding specificity and for the changes to the photophysical properties of ThT remain poorly understood. Here, we report the co-crystal structures of ThT with two alternative states of ß-2 microglobulin (ß2m); one monomeric, the other an amyloid-like oligomer. In the latter, the dye intercalates between ß-sheets orthogonal to the ß-strands. Importantly, the fluorophore is bound in such a manner that a photophysically relevant torsion is limited to a range of angles generally associated with low, not high, quantum yield. Quantum mechanical assessment of the fluorophore shows the electronic distribution to be strongly stabilized by aromatic interactions with the protein. Monomeric ß2m gives little increase in ThT fluorescence despite showing three fluorophores, at two binding sites, in configurations generally associated with high quantum yield. Our efforts fundamentally extend existing understanding about the origins of amyloid-induced photophysical changes. Specifically, the ß-sheet interface that characterizes amyloid acts both sterically and electronically to stabilize the fluorophore's ground state electronic distribution. By preventing the fluorophore from adopting its preferred excited state configuration, nonradiative relaxation pathways are minimized and quantum yield is increased.
Subject(s)
Amyloid/chemistry , Fluorescent Dyes/chemistry , Thiazoles/chemistry , beta 2-Microglobulin/chemistry , Benzothiazoles , Crystallization , Crystallography , Fluorescence , Humans , Protein Structure, SecondaryABSTRACT
Bispecific biotherapeutics offer potent and highly specific treatment options in oncology and immuno-oncology. However, many bispecific formats are prone to high levels of aggregation and instability, leading to prolonged development timelines, inefficient manufacturing, and high costs. The novel class of Mabcalin™ molecules consist of Anticalin® proteins fused to an IgG and are currently being evaluated in pre-clinical and clinical studies. Here, we describe a robust high-yield manufacturing platform for these therapeutic fusion proteins providing data up to commercially relevant scales. A platform upstream process was established for one of the Mabcalin bispecifics and then applied to other clinically relevant drug candidates with different IgG target specificities. Process performance was compared in 3â¯L bioreactors and production was scaled-up to up to 1000â¯L for confirmation. The Mabcalin proteins' structural and biophysical similarities enabled a downstream platform approach consisting of initial protein A capture, viral inactivation, mixed-mode anion exchange polishing, second polishing by cation exchange or hydrophobic interaction chromatography, viral filtration, buffer exchange and concentration by ultrafiltration/diafiltration. All three processes met their target specifications and achieved comparable clearance of impurities and product yields across scales. The described platform approach provides a fast and economic path to process confirmation and is well comparable to classical monoclonal antibody approaches in terms of costs and time to clinic.
Subject(s)
Antibodies, Monoclonal , Bioreactors , Antibodies, Monoclonal/chemistry , Chromatography , Ultrafiltration , Immunoglobulin GABSTRACT
Determination of dynamic binding capacity (DBC) for capture purification chromatographic step is usually the first experiment to be performed during downstream process development of biopharmaceuticals. In this work, we investigated the application of inline variable pathlength technology using FlowVPE for rapid determination of DBC on affinity resins for protein capture and proved its comparability with offline titer methods. This work also demonstrated that variable pathlength technology for DBC determination can be successfully applied to different classes of monoclonal antibodies and fusion proteins. This enabled rapid screening of affinity resins and optimization of the capture chromatography step. Hence, use of inline variable pathlength technology eliminated the dependency on offline titer data, traditionally used for DBC determination and accelerated overall process development timelines with less cost.
Subject(s)
Biological Products , Antibodies, Monoclonal/chemistry , Chromatography/methods , Chromatography, Affinity , TechnologyABSTRACT
The human immunodeficiency virus type 1 (HIV-1) protein Vif recruits the host E3 ubiquitin ligase, composed of cullin 5 (Cul5), Rbx2, Elongin B, and Elongin C (EloBC), to polyubiquitinate the antiviral protein APOBEC3G. Multiple regions in the C-terminal half of Vif interact with the E3 ligase. We have purified individual regions of Vif and investigated their thermodynamic contributions to the ligase assembly in vitro using isothermal titration calorimetry and fluorescence anisotropy. Our results quantify the high-affinity interactions between the Vif BC box and EloBC and between the Vif zinc finger and Cul5, as well as the modest interaction between the Vif cullin box and Cul5. Our purified Vif constructs also provide direct biochemical evidence that the Vif cullin box, containing the PPLP region, leads to the dimerization of Vif-EloBC complexes but not Cul5-Vif-EloBC complexes.
Subject(s)
HIV/metabolism , Ubiquitin-Protein Ligases/chemistry , Ubiquitin-Protein Ligases/metabolism , vif Gene Products, Human Immunodeficiency Virus/chemistry , vif Gene Products, Human Immunodeficiency Virus/metabolism , APOBEC-3G Deaminase , Amino Acid Sequence , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , HIV/genetics , Humans , Models, Molecular , Molecular Sequence Data , Multiprotein Complexes/chemistry , Multiprotein Complexes/metabolism , Protein Binding , Protein Conformation , Protein Multimerization , Thermodynamics , Ubiquitin-Protein Ligases/genetics , Ubiquitination , vif Gene Products, Human Immunodeficiency Virus/geneticsABSTRACT
Preclinical development of vaccine candidates is an important link between the discovery and manufacture of vaccines for use in human clinical trials. Here, an exploratory clinical study utilizing multiple gp120 envelope proteins as vaccine antigens was pursued, which required a harmonized platform development approach for timely and efficient manufacture of the combined HIV vaccine product. Development of cell lines, processes, and analytical methods was initiated with a transmitted founder envelope protein (CH505TF), then applied to produce three subsequent gp120 Env (envelope) variants. Cell lines were developed using the commercially available Freedom CHO DG44 kit (Life Technologies). The fed-batch cell culture production process was based on a commercially-available medium with harmonized process parameters across the variants. A platform purification process was developed utilizing a mixed mode chromatography capture step, with ceramic hydroxyapatite and ion exchange polishing steps. A suite of analytical methods was developed to establish and monitor the Quality Target Profile (QTP), release and long-term stability testing of the vaccine products. The platform development strategy was successfully implemented to produce four gp120 envelope protein variants. In some cases, minor changes to the platform were required to optimize for a particular variant; however, baseline conditions for the processes (cell line type, media & feed system, chromatography resins, and analytical approaches) remained constant, leading to successful transfer and manufacture of all four proteins in a cGMP facility. This body of work demonstrates successful pursuit of a platform development approach to manufacture important vaccine candidates and can be used as a model for other vaccine glycoproteins, such as HIV gp140 trimers or other viral glycoproteins with global health implications. Clinical trial identifier. NCT03220724, NCT03856996.
Subject(s)
AIDS Vaccines , HIV Envelope Protein gp120 , HIV Infections , Glycoproteins , HIV Antibodies , HIV Infections/prevention & control , HIV-1 , HumansABSTRACT
The ability to conduct multiple experiments in parallel significantly reduces the time that it takes to develop a manufacturing process for a biopharmaceutical. This is particularly significant before clinical entry, because process development and manufacturing are on the "critical path" for a drug candidate to enter clinical development. High-throughput process development (HTPD) methodologies can be similarly impactful during late-stage development, both for developing the final commercial process as well as for process characterization and scale-down validation activities that form a key component of the licensure filing package. This review examines the current state of the art for HTPD methodologies as they apply to cell culture, downstream purification, and analytical techniques. In addition, we provide a vision of how HTPD activities across all of these spaces can integrate to create a rapid process development engine that can accelerate biopharmaceutical drug development. Graphical Abstract.
Subject(s)
Biological Products , Biopharmaceutics , Biopharmaceutics/methods , Biopharmaceutics/trends , Cell Culture Techniques , High-Throughput Screening AssaysABSTRACT
Monoclonal antibodies (mAbs) have established themselves as the leading biopharmaceutical therapeutic modality. The establishment of robust manufacturing platforms are key for antibody drug discovery efforts to seamlessly translate into clinical and commercial successes. Several drivers are influencing the design of mAb manufacturing processes. The advent of biosimilars is driving a desire to achieve lower cost of goods and globalize biologics manufacturing. High titers are now routinely achieved for mAbs in mammalian cell culture. These drivers have resulted in significant evolution in process platform approaches. Additionally, several new trends in bioprocessing have arisen in keeping with these needs. These include the consideration of alternative expression systems, continuous biomanufacturing and non-chromatographic separation formats. This paper discusses these drivers in the context of the kinds of changes they are driving in mAb production processes.
ABSTRACT
The von Hippel-Lindau tumor suppressor protein (VHL) recruits a Cullin 2 (Cul2) E3 ubiquitin ligase to downregulate HIF-1α, an essential transcription factor for the hypoxia response. Mutations in VHL lead to VHL disease and renal cell carcinomas. Inhibition of this pathway to upregulate erythropoietin production is a promising new therapy to treat ischemia and chronic anemia. Here, we report the crystal structure of VHL bound to a Cul2 N-terminal domain, Elongin B, and Elongin C (EloC). Cul2 interacts with both the VHL BC box and cullin box and a novel EloC site. Comparison with other cullin E3 ligase structures shows that there is a conserved, yet flexible, cullin recognition module and that cullin selectivity is influenced by distinct electrostatic interactions. Our structure provides a structural basis for the study of the pathogenesis of VHL disease and rationale for the design of novel compounds that may modulate cullin-substrate receptor interactions.
Subject(s)
Transcription Factors/chemistry , Von Hippel-Lindau Tumor Suppressor Protein/chemistry , Amino Acid Sequence , Catalytic Domain , Crystallography, X-Ray , Elongin , Humans , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Quaternary , Protein Structure, SecondaryABSTRACT
The unique selectivity of mixed mode chromatography resins is driving increasing utilization of these novel selectivities into bioprocess applications. There is a need for improved fundamental understanding of protein binding to these stationary phases to enable the development of efficient and robust purification processes. A panel of four monoclonal antibodies and two model proteins were employed to characterize protein interaction with a mixed-mode chromatographic resin comprising a hydrophobic ligand with cation-exchange functionality. Binding of these proteins was studied as a function of salt concentration and pH in the presence of various mobile phase modulators. This knowledge was applied towards screening mobile phase modulators that could selectively decrease host cell protein levels during monoclonal antibody purification.