The Minimal Effect of Linker Length for Fatty Acid Conjugation to a Small Protein on the Serum Half-Life Extension

Abstract

Conjugation of serum albumin or one of its ligands (such as fatty acid) has been an effective strategy to prolong the serum half-lives of drugs via neonatal Fc receptor (FcRn)–mediated recycling of albumin. So far, fatty acid (FA) has been effective in prolonging the serum half-lives for therapeutic peptides and small proteins, but not for large therapeutic proteins. Very recently, it was reported a large protein conjugated to FA competes with the binding of FcRn with serum albumin, leading to limited serum half-life extension, because primary FA binding sites in serum albumin partially overlap with FcRn binding sites. In order to prevent such competition, longer linkers between FA and the large proteins were required. Herein, we hypothesized that small proteins do not cause substantial competition for FcRn binding to albumin, resulting in the extended serum half-life. Using a small protein (28 kDa), we investigated whether the intramolecular distance in FA-protein conjugate affects the FcRn binding with albumin and serum half-life using linkers with varying lengths. Unlike with the FA-conjugated large protein, all FA-conjugated small proteins with different linkers exhibited comparable the FcRn binding to albumin and extended serum half-life.

Protein Engineering Strategies for Improved Pharmacokinetics

“…However, the main drawbacks are the insolubility of fatty acids and their lower affinity for SA than for example antibodies and fragments thereof. [84] Moreover, the linker might be immunogenic. An immunogenic response against lipidated biopharmaceuticals has indeed been reported.…”

“…[89] Such a correlation was not observed for small proteins. [84] Bacterial Serum Albumin Binding Domains (ABDs): The second class of SA naturally binding molecules are the bacterial proteins targeting SA including Staphylococcus protein A and Streptococcus protein G. [90] The SA binding domains of these proteins, composed of about 50 amino acids (≈5 kDa) have been extensively engineered to further improve their halflife extension capability by a combination of combinatorial protein engineering, in vitro selection via phage display technology and rational design, leading to the selection of fentomolar affinity binders or of minimal size binders. [91] For example, Guo et al have fused an ABD to a human epidermal growth factor receptor 2 (HER2)-specific immunotoxin ZHER2-PE38.…”

Population pharmacokinetics of the anti-PD-1 antibody camrelizumab in patients with multiple tumor types and model-informed dosing strategy

“…10 A higher albumin concentration could be an indicator of an increased number of Fc receptor s and a related reduction in the rate of camrelizumab elimination. 20 Although albumin had a statistically significant impact on CL linear , simulation analyses demonstrated that the magnitude of its effect on camrelizumab exposure was limited ( Figure 4). As albumin levels increased from 20…”