Identification of structural origins of complex charge heterogeneity in therapeutic ACE2Fc fusion protein facilitated by free-flow isoelectric focusing

Article

作者: Zhang, Zhongli ; Shen, Pengcheng ; Cao, Ting ; Li, Hongyang ; Wu, Hai ; Wang, Lingyu ; Lu, Lihong

Fc Fusion protein represents a versatile molecular platform with considerable potential as protein therapeutics of which the charge heterogeneity should be well characterized according to regulatory guidelines. Angiotensin-converting enzyme 2 Fc fusion protein (ACE2Fc) has been investigated as a potential neutralizing agent to various coronaviruses, including the lingering SARS-CoV-2, as this coronavirus must bind to ACE2 to allow for its entry into host cells. ACE2Fc, an investigational new drug developed by Henlius (Shanghai China), has passed the Phase I clinical trial, but its huge amount of charge isoforms and complicated charge heterogeneity posed a challenge to charge variant investigation in pharmaceutical development. We employed offline free-flow isoelectric focusing (FF-IEF) fractionation, followed by detailed characterization of enriched ACE2Fc fractions, to unveil the structural origins of charge heterogeneity in ACE2Fc expressed by recombinant CHO cells. We adopted a well-tuned 3-component separation medium for ACE2Fc fractionation, the highest allowable voltage to maximize the FF-IEF separation window and a mild Protein A elution method for preservation of protein structural integrity. Through peptide mapping and other characterizations, we revealed that the intricate profiles of ACE2Fc charge heterogeneity are mainly caused by highly sialylated multi-antenna N-glycosylation. In addition, based on fraction characterization and in silico glycoprotein model analysis, we discovered that the large acidic glycans at N36, N73, and N305 of ACE2Fc were able to decrease the binding activity towards Spike (S) protein of SARS-CoV-2. Our study exemplifies the value of FF-IEF in highly complex fusion protein characterization and revealed a quantitative sialylation-activity relationship in ACE2Fc.

2023-09-01·Biochemistry. Biokhimiia

Dimeric ACE2-FC Is Equivalent to Monomeric ACE2 in the Surrogate Virus Neutralization Test.

Article

作者: Vorobiev, Ivan I ; Orlova, Nadezhda A ; Kolesov, Denis E ; Gaiamova, Elizaveta A

Angiotensin-converting enzyme 2 (ACE2) is the main cellular receptor for the dangerous sarbecoviruses SARS-CoV and SARS-CoV-2. Its recombinant extracellular domain is used to monitor the level of protective humoral immune response to a viral infection or vaccine using the surrogate virus neutralization test (sVNT). Soluble ACE2 is also considered as an option for antiviral therapy potentially insensitive to the changes in the SARS-CoV-2 spike protein. Extensive testing of the samples of patient's serum by the sVNT method requires using preparations of ACE2 or ACE2 conjugates with constant properties. We have previously obtained a cell line that is a producer of a soluble monomeric ACE2 and showed that this ACE2 variant can be used in sVNT, preferably as a conjugate with horseradish peroxidase. A cell line that generates an ACE2-Fc fusion protein with high productivity, more than 150 mg/liter of the target protein when cultured in a stirred flask, was obtained for producing a stable and universally applicable form of soluble ACE2. The affinity-purified ACE2-Fc fusion contains a mixture of dimeric and tetrameric forms, but allows obtaining linear response curves for inhibition of binding with the receptor-binding domain of the SARS-CoV-2 spike protein by antibodies. The ACE2-Fc-HRP-based sVNT testing system can be used for practical measurements of the levels of virus-neutralizing antibodies against various circulating variants of the SARS-CoV-2 virus.

2021-06-01·International immunopharmacology2区 · 医学

An overview of some potential immunotherapeutic options against COVID-19

2区 · 医学

Review

作者: Mohammadi, Mohammad Reza ; Asemani, Yahya ; Sanaei, Mahsa ; Eftekhari, Reyhaneh ; Namvarpour, Mozhdeh ; Bayat, Maryam

After the advent of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV2) in the late 2019, the resulting severe and pernicious syndrome (COVID-19) immediately was deployed all around the world. To date, despite relentless efforts to control the disease by drug repurposing, there is no approved specific therapy for COVID-19. Given the role of innate and acquired immune components in the control and elimination of viral infections and inflammatory mutilations during SARS-CoV2 pathogenesis, immunotherapeutic strategies appear to be beneficent. Passive immunotherapies such as convalescent plasma, which has received much attention especially in severe cases, as well as suppressing inflammatory cytokines, interferon administration, inhibition of kinases and complement cascade, virus neutralization with key engineered products, cell-based therapies, immunomodulators and anti-inflammatory drugs are among the key immunotherapeutic approaches to deal with COVID-19, which is discussed in this review. Also, details of leading COVID-19 vaccine candidates as the most potent immunotherapy have been provided. However, despite salient improvements, there is still a lack of completely assured vaccines for universal application. Therefore, adopting proper immunotherapies according to the cytokine pattern and involved immune responses, alongside engineered biologics specially ACE2-Fc to curb SARS-CoV2 infection until achieving a tailored vaccine is probably the best strategy to better manage this pandemic. Therefore, gaining knowledge about the mechanism of action, potential targets, as well as the effectiveness of immune-based approaches to confront COVID-19 in the form of a well-ordered review study is highly momentous.

100 项与 ACE2-Fc(University of Rhode Island) 相关的药物交易

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