A self-assembled and covalently stabilized FcRn-binding albumin fragment-payload assembly.

The short in vivo half-life of protein therapeutics remains a major limitation in their clinical translation, driving the development of human serum albumin (HSA)-based carrier systems capable of prolonged circulation through FcRn-mediated recycling. Previously, we reported an engineered HSA variant (AlbuC) that enables modular assembly with protein payloads via α-helical coiled-coil interactions; however, the reversibility of these interactions limited complex stability under diluted conditions. Here, we developed a second-generation AlbuC system by integrating heterodimeric coiled-coil motifs containing strategically positioned cysteine residues. Co-expression of the engineered AlbuC variant and a model payload in the oxidative cytoplasm of Escherichia coli SHuffle facilitated efficient in-cell heterodimerization and the formation of disulfide-stabilized AlbuC-payload complexes. Comparative biochemical analyses demonstrated that coiled-coil selection critically influenced intracellular AlbuC-payload assembly, with selected motifs yielding disulfide-linked complexes exhibiting enhanced structural stability and resistance to dilution. Importantly, the assembled complexes retained pH-dependent FcRn binding, a key feature for FcRn-mediated recycling. Furthermore, functional validation using a death receptor 5-targeting nanobody showed that a therapeutically relevant payload can be incorporated into AlbuC variants while preserving biological activity. Collectively, this study demonstrates a modular intracellular assembly strategy for generating disulfide-stabilized AlbuC-payload complexes that retain pH-dependent FcRn-binding capability and payload functionality.