Three-Dimensional Structural Characterization and Spatial Conformational Ensemble Analysis of the Ultra-Large Multivalent Fusion Protein Construct KH-002v003 (2,091 Amino Acid Residues)
Engineering extended macromolecular therapeutics requires comprehensive structural modeling to verify tertiary folding fidelity and domain accessibility across repetitive structural units. In this study, we present the structural characterization of KH-002v003, an ultra-large synthetic multivalent fusion protein construct expanding to 2,091 amino acid residues. Building upon earlier design iterations—including the 701 aa baseline framework and the 1,354–1,455 aa KH-002v002 architecture—this maximized construct integrates multiple variable heavy-chain nanobody (VHH) domains, tumor microenvironment-cleavable matrix metalloproteinase (MMP-2/9) linkers, pH-low insertion peptides (pHLIP), and C-terminal XTEN solubilization polymers. Structural predictions were executed via high-throughput homology modeling on SWISS-MODEL utilizing a 58-template ensemble superposition. Model 15, constructed against the Cryo-EM structure of the bispecific Fab-heavy chain complex (PDB ID: 8WGW.1.B, sequence identity 60.71%), yielded a peak global QMEANDisCo score of 0.58 ± 0.07. Superposition analysis revealed a dense, rigid central core dominated by antiparallel β-sheet frameworks flanked by dynamic, highly flexible loop regions. Stereochemical validation via MolProbity confirmed 92.16% of residues within favored Ramachandran regions. These findings confirm that ultra-large constructs exceeding 2,000 residues can maintain structural integrity and spatial independence for target engagement.