Toor, T., Grabowska, W.R., Johnson, A.L., Khalili, Hanieh ORCID: https://orcid.org/0000-0002-6612-1628 and Peeney, D. (2025) Histidine Tag-Specific PEGylation Improves the Circulating Half-Life of TIMP2. ACS Applied Bio Materials.

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Abstract

An overarching limitation of therapeutic biologics is the limited half-life these proteins often exhibit once in circulation. PEGylation, the chemical conjugation of proteins to poly(ethylene glycol) (PEG), is a common strategy to improve protein pharmacokinetics (PK) by enhancing stability, reducing immunogenicity, and decreasing renal clearance. Tissue Inhibitor of Metalloproteinases
2 (TIMP2) is a 22 kDa matrisome protein that exhibits therapeutic potential across a range of human disease models yet possesses a short serum halflife.
To advance the therapeutic development of recombinant His-tagged TIMP2(TIMP2), we utilized primary amine conjugation (1 kDa) and site-specific histidine conjugation (10 kDa) to improve its circulating half-life. Primary amine conjugation of PEG molecules to TIMP2 (TIMP2-a-PEG(n)) is efficient, yet it produces multiple positional isomers that are difficult to purify. Furthermore, high levels of conjugation can affect the MMP-inhibitory activity of TIMP2. Despite this, TIMP2-a-PEG(n) displays a significant improvement (11.5-fold) in serum half-life versus unconjugated TIMP2. In contrast, site-specific histidine conjugation targets the
histidine tag, enabling the purification of mono-PEGylated (TIMP2-H-PEG(1)) and di-PEGylated (TIMP2-H-PEG(2)) forms. Our findings demonstrate that TIMP2-H-PEG(1) exhibits improved PK with enhanced stability and a 6.2-fold increase in circulating halflife while maintaining MMP-inhibitory activity. These results suggest that site-specific PEGylation at a C-terminal His6 tag is a promising approach for further preclinical development of TIMP2 as a therapeutic biologic

Item Type:	Article
Identifier:	10.1021/acsabm.4c01385
Subjects:	Medicine and health > Clinical medicine > Therapeutics
Depositing User:	Hanieh Khalili
Date Deposited:	25 Feb 2025 08:28
Last Modified:	25 Feb 2025 08:30
URI:	https://repository.uwl.ac.uk/id/eprint/13277
Sustainable Development Goals:	Goal 2: Zero Hunger

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