{"doi":"10.1021/acsabm.4c01385","title":"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 His 6 tag is a promising approach for further preclinical development of TIMP2 as a therapeutic biologic.","journal":"ACS Applied Bio Materials","year":2025,"id":550221,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.949,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":1445862,"name":"Wiktoria Roksana Grabowska","orcid":null,"position":1,"is_corresponding":false},{"id":1149121,"name":"Adam L. Johnson","orcid":"0000-0002-1354-6534","position":2,"is_corresponding":false},{"id":1012258,"name":"Jane Jones","orcid":null,"position":3,"is_corresponding":false},{"id":881695,"name":"William G. Stetler‐Stevenson","orcid":"0000-0002-5500-5808","position":4,"is_corresponding":false},{"id":1445533,"name":"Hanieh Khalili","orcid":"0000-0002-6612-1628","position":5,"is_corresponding":false},{"id":881693,"name":"David Peeney","orcid":"0000-0002-9126-9923","position":6,"is_corresponding":false},{"id":1277094,"name":"Jack Toor","orcid":null,"position":0,"is_corresponding":true}],"reference_count":39,"raw_metadata":null,"created_at":"2026-07-19T02:54:16.596730Z","pmid":"39984464","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}