Pharmacokinetic, Biodistribution, and Biophysical Profiles of TNF Nanobodies Conjugated to Linear or Branched Poly(ethylene glycol)

• Published in: Bioconjugate chemistry 2012-07, Vol.23 (7), p.1452-1462
• Main Authors: Vugmeyster, Yulia, Entrican, Clifford A, Joyce, Alison P, Lawrence-Henderson, Rosemary F, Leary, Beth A, Mahoney, Christopher S, Patel, Himakshi K, Raso, Stephen W, Olland, Stephane H, Hegen, Martin, Xu, Xin
• Format: Article
• Language: English
• Subjects: Animals; Biophysics; Humans; Macaca fascicularis; Mice; Mice, Inbred Strains; Molecular Structure; Polyethylene glycol; Polyethylene Glycols - administration & dosage; Polyethylene Glycols - chemistry; Polyethylene Glycols - pharmacokinetics; Proteins; Rats; Rats, Sprague-Dawley; Single-Domain Antibodies - administration & dosage; Single-Domain Antibodies - chemistry; Tissue Distribution; Tissues; TNF inhibitors; Tumor Necrosis Factor-alpha - administration & dosage; Tumor Necrosis Factor-alpha - chemistry; Tumor Necrosis Factor-alpha - pharmacokinetics; U937 Cells
• ISSN: 1043-1802; 1520-4812
• DOI: 10.1021/bc300066a

Covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins has been used to prolong in vivo exposure of therapeutic proteins. We have examined pharmacokinetic, biodistribution, and biophysical profiles of three different tumor necrosis factor alpha (TNF) Nanobody–40 kDa PEG conjugates: linear 1 × 40 KDa, branched 2 × 20 kDa, and 4 × 10 kDa conjugates. In accord with earlier reports, the superior PK profile was observed for the branched versus linear PEG conjugates, while all three conjugates had similar potency in a cell-based assay. Our results also indicate that (i) a superior PK profile of branched versus linear PEGs is likely to hold across species, (ii) for a given PEG size, the extent of PEG branching affects the PK profile, and (iii) tissue penetration may differ between linear and branched PEG conjugates in a tissue-specific manner. Biophysical analysis (R g/R h ratio) demonstrated that among the three protein–PEG conjugates the linear PEG conjugate had the most extended time-average conformation and the most exposed surface charges. We hypothesized that these biophysical characteristics of the linear PEG conjugate accounts for relatively less optimal masking of sites involved in elimination of the PEGylated Nanobodies (e.g., intracellular uptake and proteolysis), leading to lower in vivo exposure compared to the branched PEG conjugates. However, additional studies are needed to test this hypothesis.