Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties

Soleimanpour, Saman; Hassannia, Tahereh; Motiee, Mahdieh; Amini, Abbas; Rezaee, Seyed Abdolrahim

doi:10.3109/07388551.2016.1163323

Cited by 29 publications

(23 citation statements)

References 136 publications

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“…For our vaccine platform, we chose the RBD of SARS-CoV-2 as the target antigen for the mRNA coding sequence, as described in Methods. Fusion of the IgG-Fc domain to a protein of interest has been shown to increase the half-life, immunogenicity, solubility, and delivery efficiency of the targeted protein. − Fc-fusion products are commonly used in the clinic, and 13 products have been approved by the FDA and European Medicines Agency (EMA) to date . Given the potential benefits of Fc-fusion proteins in promoting immune responses, we chose to perform the vaccination experiments with Fc fused both as mRNA and as a recombinant protein.…”

Section: Resultsmentioning

confidence: 99%

Design of SARS-CoV-2 hFc-Conjugated Receptor-Binding Domain mRNA Vaccine Delivered via Lipid Nanoparticles

et al. 2021

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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causal agent of COVID-19 and stands at the center of the current global human pandemic, with death toll exceeding one million. The urgent need for a vaccine has led to the development of various immunization approaches. mRNA vaccines represent a cell-free, simple, and rapid platform for immunization, and therefore have been employed in recent studies toward the development of a SARS-CoV-2 vaccine. Herein, we present the design of an mRNA vaccine, based on lipid nanoparticles (LNPs)-encapsulated SARS-CoV-2 human Fc-conjugated receptorbinding domain (RBD-hFc). Several ionizable lipids have been evaluated in vivo in a luciferase (luc) mRNA reporter assay, and two leading LNPs formulations have been chosen for the subsequent RBD-hFc mRNA vaccine strategy. Intramuscular administration of LNP RBD-hFc mRNA elicited robust humoral response, a high level of neutralizing antibodies and a Th1-biased cellular response in BALB/c mice. The data in the current study demonstrate the potential of these lipids as promising candidates for LNP-based mRNA vaccines in general and for a COVID19 vaccine in particular.

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Section: Resultsmentioning

confidence: 99%

Design of SARS-CoV-2 hFc-Conjugated Receptor-Binding Domain mRNA Vaccine Delivered via Lipid Nanoparticles

et al. 2021

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“… 59 , 60 Glycoproteins are generally folded and soluble, 61 but fusion partners or other tags can be added if target solubility is an issue (e.g., maltose-binding protein fusions) 62 , 63 or dimeric presentation is desired. 64 While we used the G. princeps derived SP for enhanced secretion, 46 SP choice is likely to be an empirical parameter for each recombinant protein and cell system, 65 and alternative SPs may significantly increase secretion and recovery rates. 65 , 66 As we employed small-scale expressions to obtain multiple ERBB constructs, standard losses from each step (e.g., buffer exchange) may also have resulted in suboptimal yields.…”

Section: Resultsmentioning

confidence: 99%

“…While we present this expression/purification scheme using RTK ECDs as exemplars, it can be expanded to other heavily glycosylated and disulfide-dependent proteins and modified for specific post-translational characteristics. For example, if a specific glyco-pattern is desired, other cell types (e.g., CHO) can be used produce different glycosylation patterns. , Glycoproteins are generally folded and soluble, but fusion partners or other tags can be added if target solubility is an issue (e.g., maltose-binding protein fusions) , or dimeric presentation is desired . While we used the G.…”

Section: Resultsmentioning

confidence: 99%

Mammalian Expression and In Situ Biotinylation of Extracellular Protein Targets for Directed Evolution

et al. 2020

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Directed evolution is a powerful tool for the selection of functional ligands from molecular libraries. Extracellular domains (ECDs) of cell surface receptors are common selection targets for therapeutic and imaging agent development. Unfortunately, these proteins are often post-translationally modified and are therefore unsuitable for expression in bacterial systems. Directional immobilization of these targets is further hampered by the absence of biorthogonal groups for site-specific chemical conjugation. We have developed a nonadherent mammalian expression system for rapid, high-yield expression of biotinylated ECDs. ECDs from EGFR, HER2, and HER3 were site-specifically biotinylated in situ and recovered from the cell culture supernatant with yields of up to 10 mg/L at >90% purity. Biotinylated ECDs also contained a protease cleavage site for rapid and selective release of the ECD after immobilization on avidin/streptavidin resins and library binding. A model mRNA display selection round was carried out against the HER2 ECD with the HER2 affibody expressed as an mRNA–protein fusion. HER2 affibody–mRNA fusions were selectively released by thrombin and quantitative PCR revealed substantial improvements in the enrichment of functional affibody–mRNA fusions relative to direct PCR amplification of the resin-bound target. This methodology allows rapid purification of high-quality targets for directed evolution and selective elution of functional sequences at the conclusion of each selection round.

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“…The importance of recombinant Fc-fusion proteins is inevitably dependent on their biophysical, biochemical and pharmacological characteristics, which exist in the constant region of the Fc fragment. Many medications with the FDA approved products are available for many different ranges of medical intervention (Czajkowsky et al 2012;Soleimanpour et al 2017). Furthermore, attempts for making appropriate vaccines against infectious diseases such as Ebola (Konduru et al 2011), TB (Soleimanpour et al 2015;Farsiani et al 2016;Mosavat et al 2016), In uenza (Loureiro et al 2011) and SARS-CoV-2 (Ren et al 2020) are still ongoing.…”

Section: Discussionmentioning

confidence: 99%

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as a Recombinant Fc Fusion Multi-Stage Vaccine Candidate Against Mycobacterium Tuberculosis

Karbalaei

Mosavat

Soleimanpour

et al. 2020

Preprint

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AimsTuberculosis (TB) is one of the life-threatening infectious diseases, caused by Mycobacterium tuberculosis (M.tb). In the present study, a multi-stage M.tb immunodominant Fcγ1 fusion protein (Ag85B:HspX:hFcγ1) was produced and its immunogenicity as an hFcγRI targeted delivery systems for selective antigen presentation was evaluated in a mouse model. Methods and ResultsThe novel Ag85B:HspX:hFcγ1 recombinant fusion protein was designed and expressed in the Pichia pastoris (P. pastoris). After affinity chromatography purification, the purity of Ag85B:HspX:hFcγ1 was confirmed by ELISA, SDS-PAGE, and Western blotting methods. The immunogenicity of the construct was evaluated by assessing interferon-γ (IFN-γ) and transforming growth factor-beta (TGF-β) in a mouse model. Co-localization results of Ag85B:HspX:hFcγ1 with hFcγRI (CD64) confirmed its function for binding with its receptor and inducing Th1 selective responses. There was a significant difference in the expression of both IFN-γ, (P≤0.02) and TGF-β, (P=0.05).ConclusionsThe co-localization assay confirmed functionally the binding of the Ag85B:HspX:hFcγ1 to CD64 (FcγRI). Furthermore, in vitro assay showed that Ag85B:HspX:hFcγ1 can stimulate a modulated immune response in favor of anti-intracellular microbes, as IFN-γ increased, and also TGF-β as an immune-modulatory cytokine prevented the induction of hypersensitivity reactions.Significance and Impact of StudyThe combination of Ag85B as the most immunodominant M.tb Ag with HspX, as an Ag and adjuvant, could open a new venue for more studies for the design of multi-stage subunit vaccines for TB. Of note, an Fcγ1 fusion protein can be considered as a functional approved selective delivery vehicle for targeting antigen-presenting cells (APCs) and inducing cross-presentation.

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Fcγ1 fragment of IgG1 as a powerful affinity tag in recombinant Fc-fusion proteins: immunological, biochemical and therapeutic properties

Cited by 29 publications

References 136 publications

Design of SARS-CoV-2 hFc-Conjugated Receptor-Binding Domain mRNA Vaccine Delivered via Lipid Nanoparticles

Design of SARS-CoV-2 hFc-Conjugated Receptor-Binding Domain mRNA Vaccine Delivered via Lipid Nanoparticles

Mammalian Expression and In Situ Biotinylation of Extracellular Protein Targets for Directed Evolution

Production and Evaluation of Ag85B:HspX:hFcγ1 Immunogenicity as a Recombinant Fc Fusion Multi-Stage Vaccine Candidate Against Mycobacterium Tuberculosis

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