AlbuCatcher for Long-Acting Therapeutics

Abstract

Therapeutic proteins, pivotal for treating diverse human diseases due to their biocompatibility and high selectivity, often face challenges such as rapid serum clearance, enzymatic degradation, and immune responses. To address these issues and enable prolonged therapeutic efficacy, techniques to extend the serum half-life of therapeutic proteins are crucial. The AlbuCatcher, a conjugate of human serum albumin (HSA) and SpyCatcher, was proposed as a general technique to extend the serum half-life of diverse therapeutic proteins. HSA, the most abundant blood protein, exhibits a long intrinsic half-life through Fc receptor (FcRn)-mediated recycling. The SpyTag/SpyCatcher (ST/SC) system, known for forming irreversible isopeptide bonds, was employed to conjugate HSA and therapeutic proteins. Site-specific HSA conjugation to SC was achieved using an inverse electron-demand Diels-Alder (IEDDA) reaction, minimizing activity loss. Using urate oxidase (Uox) as a model protein with a short half-life, the small ST was fused to generate Uox-ST. Then, HSA-conjugated Uox (Uox-HSA) was successfully prepared via the Uox-ST/AlbuCatcher reaction. In vitro enzyme assays demonstrated that the impact of ST fusion and HSA conjugation on Uox enzymatic activity is negligible. Pharmacokinetics studies in mice revealed that Uox-HSA exhibits a significantly longer serum half-life (about 18 h) compared to Uox-WT (about 2 h). This extended half-life is attributed to FcRn-mediated recycling of HSA-conjugated Uox, demonstrating the effectiveness of the AlbuCatcher strategy in enhancing the pharmacokinetics of therapeutic proteins.

Figure 1

Schematic illustration showing (A) the interaction between HSA (yellow) and FcRn (green) (PDB ID: 4N0F) with the free cysteine at position 34 on HSA marked red, (B) the interaction between ST (orange) and SC (blue) with the side chains of the frTet incorporation sites (S50, D83, T89, E97, and H113) highlighted in red, (C) the reactions—inverse electron-demand Diels–Alder reaction (IEDDA) and SpyTag/SpyCatcher system (ST/SC)—and (D) the construction of AlbuCatcher and its reaction with ST-fused urate oxidase (Uox) to obtain HSA-conjugated Uox.

Figure 2

Preparation and characterization of SC variants (wild-type SC (WT), SC-D83 (D83), SC-H113 (H113), SC-E97 (E97), SC-T89 (T89), and SC-S50 (S50)). (A) Coomassie brilliant blue-stained protein gels of purified SC variants. Lane MW: molecular weight marker. (B) MALDI-TOF MS analysis of intact SC-WT, SCD83, and SC-T89. (C) Fluorescence analysis (illumination λex = 302 nm, with wavelengths at 510 to 610 nm in Chemidoc XRS + system) and Coomassie brilliant blue-stained protein gels of SC variants incubated with (+) or without (−) TCO-Cy3. Lane MW: molecular weight marker.

Figure 3

Preparation and characterization of AlbuCatcher. (A) SDS-PAGE analysis of the reaction mixture of SC-frTet variants in the absence (−) or presence (+) of HSA-TCO. (B) Protein gel of purification stained with Coomassie brilliant blue. Lane 1, SC-T89; Lane 2, HSA/HSA-TCO; Lane 3, reaction mixture; Lane 4, flow-through; Lane 5, washed solution; Lane 6, eluted solution; Lane 7, column-purified AlbuCatcher. (C) MALDI-TOF mass spectrum of AlbuCatcher.

Figure 4

Purification and characterization of Uox species (Uox-WT, Uox-ST, and Uox-HSA). (A) Coomassie brilliant blue-stained protein gel: Lane 1, Uox-WT; Lane 2, Uox-ST. (B) MALDI-TOF analyses of Uox-WT and Uox-ST. (C) The generation and purification of the Uox-HSA conjugate: Lane 1, Uox-ST; Lane 2, AlbuCatcher, Lane 3, reaction mixture; Lane 4, column-purified Uox-HSA; Lane 5, residual AlbuCatcher. (D) MALDI-TOF MS of the Uox-HSA conjugate.

Figure 5

Enzymatic assay of Uox variants in vitro and in vivo. (A) The relative enzymatic activity of the Uox species. The relative enzymatic activities were normalized to the enzymatic activity of Uox-WT. Experiments were performed in triplets, and error bars mean standard deviations. The data were subjected to one-way ANOVA with Tukey’s posthoc test, and “ns” means there is no significant difference. (B) Pharmacokinetic studies of Uox-WT and Uox-HSA conjugate in mice (n = 5). Samples were intravenously injected into BALB/c female mice. The remaining enzymatic activity of the residual Uox variants were measured at different time points: 0.25, 1.5, and 3 h for Uox-WT; 0.25, 1.5, 3, 6, 12, 24, 36, and 48 h for Uox-HSA conjugate.