Integrating Tumor Targeting and Half-Life Extension into a Dual-Surfaced Single-Domain ADAPT Protein

Folate receptor alpha (FRα) is frequently overexpressed in ovarian cancer while showing limited expression in healthy tissues. Targeting of FRα has been extensively investigated using antibody-based therapies but less so with small scaffold proteins. Small scaffold proteins represent an alternative approach with potential advantages such as site-specific conjugation and deep tumor penetration but are limited by a short half-life. Here, we describe the development of a dual-surfaced ADAPT scaffold protein (6 kDa) with simultaneous bispecificity for FRα and albumin. Using combinatorial libraries and phage display, we identified a candidate, termed ADAPT22, with high isoform selectivity and subnanomolar affinity for FRα (KD = 0.8 nM), paired with a low nanomolar affinity for albumin (KD = 2.4 nM). Importantly, ADAPT22 is able to interact with both targets simultaneously, truly combining extended retention in blood via albumin association and tumor targeting via FRα within a single-domain protein. The simultaneous nature was verified in vitro to soluble proteins and to FRα-positive cells and in vivo using a mouse xenograft model. In mice, ADAPT22 demonstrated markedly extended blood retention, reduced renal uptake, and specific accumulation in FRα-positive tumors, confirming simultaneous dual-surface binding. To the best of our knowledge, this is the smallest half-life extended affinity protein designed for therapeutic targeting of FRα.