Reteplase Fc-fusions produced in N. benthamiana are able to dissolve blood clots ex vivo.

Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE

Original Publication: San Francisco, CA : Public Library of Science

Fibrinolytic Agents*/chemistry ; Fibrinolytic Agents*/pharmacology ; Immunoglobulin Fc Fragments*/biosynthesis ; Immunoglobulin Fc Fragments*/chemistry ; Immunoglobulin Fc Fragments*/genetics ; Immunoglobulin Fc Fragments*/pharmacology ; Recombinant Fusion Proteins*/biosynthesis ; Recombinant Fusion Proteins*/chemistry ; Recombinant Fusion Proteins*/pharmacology ; Tissue Plasminogen Activator*/biosynthesis ; Tissue Plasminogen Activator*/chemistry ; Tissue Plasminogen Activator*/genetics ; Tissue Plasminogen Activator*/pharmacology; Fibrinolysis/*drug effects ; Nicotiana/*genetics; Humans ; Recombinant Proteins/biosynthesis ; Recombinant Proteins/chemistry ; Recombinant Proteins/genetics ; Recombinant Proteins/pharmacology ; Nicotiana/metabolism

Thrombolytic and fibrinolytic therapies are effective treatments to dissolve blood clots in stroke therapy. Thrombolytic drugs activate plasminogen to its cleaved form plasmin, a proteolytic enzyme that breaks the crosslinks between fibrin molecules. The FDA-approved human tissue plasminogen activator Reteplase (rPA) is a non-glycosylated protein produced in E. coli. rPA is a deletion mutant of the wild-type Alteplase that benefits from an extended plasma half-life, reduced fibrin specificity and the ability to better penetrate into blood clots. Different methods have been proposed to improve the production of rPA. Here we show for the first time the transient expression in Nicotiana benthamiana of rPA fused to the immunoglobulin fragment crystallizable (Fc) domain on an IgG1, a strategy commonly used to improve the stability of therapeutic proteins. Despite our success on the expression and purification of dimeric rPA-Fc fusions, protein instability results in high amounts of Fc-derived degradation products. We hypothesize that the "Y"- shape of dimeric Fc fusions cause steric hindrance between protein domains and leads to physical instability. Indeed, mutations of critical residues in the Fc dimerization interface allowed the expression of fully stable rPA monomeric Fc-fusions. The ability of rPA-Fc to convert plasminogen into plasmin was demonstrated by plasminogen zymography and clot lysis assay shows that rPA-Fc is able to dissolve blood clots ex vivo. Finally, we addressed concerns with the plant-specific glycosylation by modulating rPA-Fc glycosylation towards serum-like structures including α2,6-sialylated and α1,6-core fucosylated N-glycans completely devoid of plant core fucose and xylose residues.
Competing Interests: The authors have declared that no competing interests exist.

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P 31131 Austria FWF_ Austrian Science Fund FWF

0 (Fibrinolytic Agents)
0 (Immunoglobulin Fc Fragments)
0 (Recombinant Fusion Proteins)
0 (Recombinant Proteins)
DQA630RIE9 (reteplase)
EC 3.4.21.68 (Tissue Plasminogen Activator)

Date Created: 20211130 Date Completed: 20220105 Latest Revision: 20231213

20231215

PMC8631678

10.1371/journal.pone.0260796

34847186