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

Abstract

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.

Influence of protein properties and protein modification on biodistribution and tumor uptake of anticancer antibodies, antibody derivatives, and non‐Ig scaffolds

“…Like chain length, the shape of PEG chains can influence the effect of PEGylation on circulation time of PEGylated proteins. Despite the fact that total PEG length was similar, the use of branched PEG chains (2 × 20 or 4 × 10 kDa) increased the serum half‐life of tumor necrosis factor‐alpha targeting VHH more than PEGylation with linear PEG chains (1 × 40 kDa), as studied in mice, rats, and cynomolgus monkeys …”

Site-Specific Functionalization of Proteins and Their Applications to Therapeutic Antibodies

“…Several branched and non-branched PEG structures have been evaluated for the effect on renal clearance. Enhanced PK profiles for branched PEG conjugates have consistently been described for therapeutic proteins in the literature [112–114]. Recently for instance, single domain antibodies labeled with 2x20 kDa PEG were shown to be superior over 1x40 kDa and 4x10 kDa labeling without affecting the biological activity [114].…”

“…Enhanced PK profiles for branched PEG conjugates have consistently been described for therapeutic proteins in the literature [112–114]. Recently for instance, single domain antibodies labeled with 2x20 kDa PEG were shown to be superior over 1x40 kDa and 4x10 kDa labeling without affecting the biological activity [114]. Biodistribution studies showed higher serum exposure of the antibody, though this was not the case for some tissues.…”

“…Interestingly, the production of hyperglycosylated antibodies fragments in engineered cell lines demonstrated longer half-lives as well as reduced proteolysis [109]. Alternatively, Fab fragments conjugated to PEG benefits, beside from the increased size, also from the reduced intracellular uptake and proteolytic degradation by masking sensitive sites [114]. …”

Absorption, Distribution, Metabolism, and Excretion (ADME) Studies of Biotherapeutics for Autoimmune and Inflammatory Conditions

“…Yamoka et al showed differences in tissue uptake for naked (non-protein attached) PEG molecules of different molecular weight after IV administration in mice (42,43). A recent case study with a PEGylated TNF-binding Nanobody suggested that the impact of the degree of PEG branching on tissue distribution may not necessarily parallel the impact on serum PK (48). In this study, mice were given a single IV injection of 125 I-labeled anti-TNF Nanobody conjugated to either linear or branched 40-kDa PEG and serum PK, and tissue penetration in normal mice was examined.…”