IN SILICO DEVELOPMENT AND CHARACTERIZATION OF SP2 ANTIBODY MUTANTS FOR IMPROVED ADC TARGETING GALECTIN-3BP

Cancer comprises a group of diseases characterized by irregular cell growth and proliferation. In this context, the Galectin-3 Binding Protein (Gal-3BP), a highly glycosylated protein, has been explored in relation to neoplastic progression, immune evasion, proliferative signaling, angiogenesis, and metastasis. These attributes make it a promising therapeutic target, particularly for antibody-drug conjugate (ADC) therapies. Advances in targeting Gal-3BP have led to the development of an ADC using the SP2 antibody, which has demonstrated efficacy in in vitro assays. ADCs are an effective strategy for delivering cytotoxic payloads to tumor cells while minimizing damage to healthy tissues. The SP2 antibody binds near a carbohydrate on Gal-3BP, which varies depending on the cellular context. Therefore, creating SP2 mutants that avoid carbohydrate binding offers several benefits, including increased specificity for the protein target, more consistent therapeutic responses, and enhanced stability. These modifications aim to improve the efficacy and safety of ADCs, making treatments more reliable and adaptable across different cellular environments. The objective is to create optimized antibody mutants that leverage these benefits to improve therapeutic outcomes in cancer treatment. We modeled the Gal-3BP and the SP2 Fv region to obtain their 3D structures. Our mutational routine was performed using a custom pipeline developed by our group. Molecular docking was then employed to identify candidate poses for the complex. Subsequently, we assessed the stability and flexibility of these complexes through heated molecular dynamics simulations. Our results identified several promising candidates for further investigation. Docking analysis successfully isolated mutants with high specificity for the target epitope. For these selected candidates, molecular dynamics simulations confirmed stable structures and appropriate conformations for protein binding. The stability of the CDRs was essential for antibody interactions, largely maintained by hydrophobic interactions. These in silico findings will be further validated through subsequent experimental analyses.