Structural and functional characterization of a single chain diabody targeting hERG1/beta1 integrin complex in cancer

Cancer progression is driven not only by genetic alterations but also by dysregulated signaling pathways involving ion channels and adhesion receptors. Among these, the voltage-gated potassium channel hERG1 (Kv11.1) and the β1 integrin subunit form a tumor-specific macromolecular complex that contributes to proliferation, survival, migration, and therapy resistance in several malignancies, including pancreatic ductal adenocarcinoma (PDAC) and triple-negative breast cancer (TNBC). Due to its restricted expression pattern and functional relevance, the hERG1/β1 complex represents a promising diagnostic and therapeutic target. This PhD project aimed to characterize the structural and biophysical properties of the hERG1/β1 complex and to evaluate innovative recombinant antibody formats capable of selectively targeting this assembly. First, structural analyses were conducted using FSEC, co-immunoprecipitation, and fluorescence-tagged constructs to determine whether a truncated hERG1-TS variant could maintain interaction with endogenous β1 integrin and to explore how different stimuli (ligand binding, adhesion, antibody activation) modulate complex formation. Parallel efforts focused on cloning fluorescent β1-mCherry variants and optimizing conditions for structural studies. A second major objective was the structural and biophysical characterization of a bispecific single-chain diabody (scDb-hERG1/β1) engineered to simultaneously bind hERG1 and β1 integrin. Crystallization trials and SPR assays were performed to investigate stability, ligand affinity, and binding kinetics. The scDb format was also explored as a platform for functional derivatives, including a TRAIL-fused trispecific construct and imaging-compatible labeled versions. Finally, the antitumor potential of scDb-based therapeutics was assessed in PDAC and TNBC models. Functional assays revealed that scDb-hERG1/β1 and its derivatives reduce cell viability and enhance apoptosis. Combination treatments demonstrated synergistic effects with gemcitabine in PDAC cells and with statins—drugs with emerging anticancer activity. In TNBC models, the trispecific scDb-TRAIL construct showed selective cytotoxicity toward malignant cells while sparing non-transformed counterparts. Overall, this work provides new structural insights into a tumor-specific hERG1/β1 signaling platform and supports the development of antibody-based strategies targeting ion channel–receptor complexes. These findings highlight the hERG1/β1 complex as a valuable biomarker and a promising therapeutic entry point for precision oncology.