Histidine Tag-Specific PEGylation Improves the Circulating Half-Life of TIMP2.

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 half-life. 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 half-life 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.