Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy.
Ontology highlight
ABSTRACT:
SUBMITTER: Rosier BJHM
PROVIDER: S-EPMC5708335 | biostudies-literature | 2017 Jun
REPOSITORIES: biostudies-literature
Publications
Incorporation of native antibodies and Fc-fusion proteins on DNA nanostructures via a modular conjugation strategy.
Rosier Bas J H M BJHM Cremers Glenn A O GAO Engelen Wouter W Merkx Maarten M Brunsveld Luc L de Greef Tom F A TFA
Chemical communications (Cambridge, England) 20170601 53
A photocrosslinkable protein G variant was used as an adapter protein to covalently and site-specifically conjugate an antibody and an Fc-fusion protein to an oligonucleotide. This modular approach enables straightforward decoration of DNA nanostructures with complex native proteins while retaining their innate binding affinity, allowing precise control over the nanoscale spatial organization of such proteins for in vitro and in vivo biomedical applications. ...[more]
Similar Datasets
Project description:Therapeutic antibodies have captured substantial attention due to the relatively high rate at which these products reach marketing approval, and the subsequent commercial success they frequently achieve. In the 2000s, a total of 20 antibodies (18 full-length IgG and 2 Fab) were approved by the Food and Drug Administration (FDA) or European Medicines Agency (EMA). In the 2010s to date, an additional 3 antibodies (denosumab, belimumab, ipilimumab) have been approved and one antibody-drug conjugate (brentuximab vedotin) is undergoing regulatory review and may be approved in the US by August 30, 2011. However, a less heralded group of antibody-based therapeutics comprising proteins or peptides fused with an Fc is following the success of classical antibodies.
| S-EPMC3225844 | biostudies-literature
Project description:Multivalent display of receptor-engaging antibodies or ligands can enhance their activity. Instead of achieving multivalency by attachment to preexisting scaffolds, here we unite form and function by the computational design of nanocages in which one structural component is an antibody or Fc-ligand fusion and the second is a designed antibody-binding homo-oligomer that drives nanocage assembly. Structures of eight nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage, respectively, closely match the corresponding computational models. Antibody nanocages targeting cell surface receptors enhance signaling compared with free antibodies or Fc-fusions in death receptor 5 (DR5)-mediated apoptosis, angiopoietin-1 receptor (Tie2)-mediated angiogenesis, CD40 activation, and T cell proliferation. Nanocage assembly also increases severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-angiotensin-converting enzyme 2 (ACE2) fusion proteins.
| S-EPMC8592034 | biostudies-literature
Project description:Antibodies are widely used in biology and medicine, and there has been considerable interest in multivalent antibody formats to increase binding avidity and enhance signaling pathway agonism. However, there are currently no general approaches for forming precisely oriented antibody assemblies with controlled valency. We describe the computational design of two-component nanocages that overcome this limitation by uniting form and function. One structural component is any antibody or Fc fusion and the second is a designed Fc-binding homo-oligomer that drives nanocage assembly. Structures of 8 antibody nanocages determined by electron microscopy spanning dihedral, tetrahedral, octahedral, and icosahedral architectures with 2, 6, 12, and 30 antibodies per nanocage match the corresponding computational models. Antibody nanocages targeting cell-surface receptors enhance signaling compared to free antibodies or Fc-fusions in DR5-mediated apoptosis, Tie2-mediated angiogenesis, CD40 activation, and T cell proliferation; nanocage assembly also increases SARS-CoV-2 pseudovirus neutralization by α-SARS-CoV-2 monoclonal antibodies and Fc-ACE2 fusion proteins. We anticipate that the ability to assemble arbitrary antibodies without need for covalent modification into highly ordered assemblies with different geometries and valencies will have broad impact in biology and medicine.
| S-EPMC7724662 | biostudies-literature
Project description:The synthesis of antibody-oligonucleotide conjugates has enabled the development of highly sensitive bioassays for specific epitopes in the laboratory and clinic. Most synthetic schemes to generate these hybrid molecules require expensive reagents, significant quantities of input antibody, and multistep purification routes; thus limiting widespread application. Herein a facile and robust conjugation strategy is reported that involves "plug-and-play" antibody conjugation with succinimidyl-functionalized oligonucleotides, which are high yielding and compatible for use directly after buffer exchange. The succinimidyl-linked oligonucleotides are synthesized with 5'-amine-modified oligonucleotides and disuccinimidyl suberate (DSS), both of which are inexpensive and commercially available. Direct incubation of the resulting stable succinimidyl- oligonucleotide conjugates with commercial antibodies yields conjugates ready for use after benchtop buffer exchange. It is demonstrated that the resulting oligonucleotide-antibody and oligonucleotide-streptavidin conjugates retain potent and specific binding in activity-dependent proximity ligation imaging, and proximity ligation-mediated qPCR detection of endogenous proteins in native cellular contexts down to picogram levels of whole proteome. This DSS conjugation strategy should be widely applicable in the synthesis of protein-oligonucleotide conjugates.
| S-EPMC6773266 | biostudies-literature
Project description:Since the first description in 1989 of CD4-Fc-fusion antagonists that inhibit human immune deficiency virus entry into T cells, Fc-fusion proteins have been intensely investigated for their effectiveness to curb a range of pathologies, with several notable recent successes coming to market. These promising outcomes have stimulated the development of novel approaches to improve their efficacy and safety, while also broadening their clinical remit to other uses such as vaccines and intravenous immunoglobulin therapy. This increased attention has also led to non-clinical applications of Fc-fusions, such as affinity reagents in microarray devices. Here we discuss recent results and more generally applicable strategies to improve Fc-fusion proteins for each application, with particular attention to the newer, less charted areas.
| S-EPMC3491832 | biostudies-literature
Project description:Anisotropic gold nanorods provide a convenient combination of properties, such as tunability of plasmon resonances and strong extinction cross sections in the near-infrared to red spectral region. These properties have created significant interest in the development of antibody conjugation methods for synthesis of targeted nanorods for a number of biomedical applications, including molecular specific imaging and therapy. Previously published conjugation approaches have achieved molecular specificity. However, the current conjugation methods have several downsides including low stability and potential cytotoxicity of bioconjugates that are produced by electrostatic interactions, as well as lack of control over antibody orientation during covalent conjugation. Here we addressed these shortcomings by introducing directional antibody conjugation to the gold nanorod surface. The directional conjugation is achieved through the carbohydrate moiety, which is located on one of the heavy chains of the Fc portion of most antibodies. The carbohydrate is oxidized under mild conditions to a hydrazide reactive aldehyde group. Then, a heterofunctional linker with hydrazide and dithiol groups is used to attach antibodies to gold nanorods. The directional conjugation approach was characterized using electron microscopy, zeta potential, and extinction spectra. We also determined spectral changes associated with nanorod aggregation; these spectral changes can be used as a convenient quality control of nanorod bioconjugates. Molecular specificity of the synthesized antibody targeted nanorods was demonstrated using hyperspectral, optical and photoacoustic imaging of cancer cell culture models. Additionally, we observed characteristic changes in optical spectra of molecular specific nanorods after their interactions with cancer cells; the observed spectral signatures can be explored for sensitive cancer detection.
| S-EPMC3891905 | biostudies-literature
Project description:Fusion proteins combining hexavalent TRAIL with antibody fragments allow for a targeted delivery and efficient apoptosis induction in tumor cells. Here, we analyzed scFv-Fc-scTRAIL molecules directed against EGFR, HER2, HER3, and EpCAM as well as an untargeted Fc-scTRAIL fusion protein for their potentials to induce cell death both in vitro and in a xenograft tumor model in vivo. The scFv-Fc-scTRAIL fusion protein directed against EGFR as well as the fusion protein directed against EpCAM showed targeting effects on the two tested colorectal carcinoma cell lines Colo205 and HCT116, while a fusion protein targeting HER3 was more effective than untargeted Fc-scTRAIL only on Colo205 cells. Interestingly, another anti-HER3 scFv-Fc-scTRAIL fusion protein exhibiting approximately 10-fold weaker antigen binding as well as the HER2-directed molecule were unable to increase cytotoxicity compared to Fc-scTRAIL. A comparison of EC50 values of cell death induction and antigen binding supports the assumption that high affinity antigen binding is one of the requirements for in vitro targeting effects. Furthermore, a minimal number of expressed target antigens might be required for increased cytotoxicity of targeted compared to non-targeted molecules. In a Colo205 s.c. xenograft tumor model, strongest antitumor activity was observed for the anti-HER3 scFv-Fc-scTRAIL fusion protein based on antibody 3-43, with complete tumor remissions after six twice-weekly injections. Surprisingly, a similar in vivo activity was also observed for untargeted Fc-scTRAIL in this tumor model, indicating that additional factors contribute to the potent efficacy of targeted as well as untargeted hexavalent Fc-scTRAIL fusion proteins in vivo.
| S-EPMC5834252 | biostudies-literature
Project description:The success of Fc-fusion bio-therapeutics has spurred the development of other Fc-fusion products for treating and/or vaccinating against a range of diseases. We describe a method to modulate their function by converting them into well-defined stable polymers. This strategy resulted in cylindrical hexameric structures revealed by tapping mode atomic force microscopy (AFM). Polymeric Fc-fusions were significantly less immunogenic than their dimeric or monomeric counterparts, a result partly owing to their reduced ability to interact with critical Fc-receptors. However, in the absence of the fusion partner, polymeric IgG1-Fc molecules were capable of binding selectively to Fc?Rs, with significantly increased affinity owing to their increased valency, suggesting that these reagents may prove of immediate utility in the development of well-defined replacements for intravenous immunoglobulin (IVIG) therapy. Overall, these findings establish an effective IgG Fc-fusion based polymeric platform with which the therapeutic and vaccination applications of Fc-fusion immune-complexes can now be explored.
| S-EPMC3216605 | biostudies-literature
Project description:Manufacturing of biopharmaceuticals involves recombinant protein expression in host cells followed by extensive purification of the target protein. Yet, host cell proteins (HCPs) may persist in the final drug product, potentially reducing its quality with respect to safety and efficacy. Consequently, residual HCPs are closely monitored during downstream processing by techniques such as enzyme-linked immunosorbent assay (ELISA) or high-performance liquid chromatography combined with tandem mass spectrometry (HPLC-MS/MS). The latter is especially attractive as it provides information with respect to protein identities. Although the applied HPLC-MS/MS methodologies are frequently optimized with respect to HCP identification, acquired data is typically analyzed using standard settings. Here, we describe an improved strategy for evaluating HPLC-MS/MS data of HCP-derived peptides, involving probabilistic protein inference and peptide detection in the absence of fragment ion spectra. This data analysis workflow was applied to data obtained for drug products of various biotherapeutics upon protein A affinity depletion. The presented data evaluation strategy enabled in-depth comparative analysis of the HCP repertoires identified in drug products of the monoclonal antibodies rituximab and bevacizumab, as well as the fusion protein etanercept. In contrast to commonly applied ELISA strategies, the here presented workflow is process-independent and may be implemented into existing HPLC-MS/MS setups for drug product characterization and process development. Graphical abstract.
| S-EPMC7442769 | biostudies-literature
Project description:Antibody dependent (AD) functions such as AD cellular cytotoxicity (ADCC) were associated with lower viral load (VL) in untreated HIV progressors and protection from HIV infection in the modestly protective RV144 HIV vaccine trial. Target cells used to measure ADCC, AD complement deposition (ADCD), and AD cellular trogocytosis (ADCT) have been either HIV envelope (Env) gp120-coated CEM.NKr.CCR5 cells or HIV infected cell cultures. In HIV infected cell cultures, uninfected bystander cells take up gp120 shed from infected cells. Both gp120-coated and gp120+ bystander cells expose CD4 induced (CD4i) epitopes, which are normally hidden in native trimeric Env expressed by genuinely HIV infected cells since Nef and Vpu downmodulate cell surface CD4. Antibody dependent assays using either of these target cells probe for CD4i Abs that are abundant in HIV+ plasma but that do not recognize HIV-infected cells. Here, we examined ADCC, ADCD, and ADCT functions using a target cell line, sorted HIV-infected cell line cells, whose HIV infection frequency nears 100% and that expresses HIV Env in a native trimeric closed conformation. Using sorted HIV-infected cells (siCEM) as targets, we probed the binding and AD functions of anti-gp120/Env Abs in plasma from HIV-infected untreated progressor (UTP, n = 18) and treated (TP, n = 24) subjects, compared to that in Elite controllers (EC, n = 37) and Viral Controllers (VC, n = 16), which are rare subsets of HIV-infected individuals who maintain undetectable or low VL, respectively, without treatment. Gp120-coated beads were used to measure AD cellular phagocytosis. Equivalent concentrations of input IgG in plasma from UTPs, ECs, and VCs supported higher levels of all AD functions tested than plasma from TPs. When AD activities were normalized to the concentration of anti-gp120/Env-specific Abs, between-group differences largely disappeared. This finding suggests that the anti-gp120/Env Abs concentrations and not their potency determined AD functional levels in these assays. Elite controllers did differ from the other groups by having AD functions that were highly polyfunctional and highly correlated with each other. PCR measurement of HIV reservoir size showed that ADCC activity was higher in ECs and VCs with a reservoir size below the limit of detection compared to those having a measurable HIV reservoir size.
| S-EPMC7555699 | biostudies-literature