Novel Method for Preparation of Site-Specific, Stoichiometric-Controlled Dual Warhead Conjugate of FGF2 via Dimerization Employing Sortase A-Mediated Ligation.

Targeted therapies are rapidly evolving modalities of cancer treatment. The largest group of currently developed biopharmaceuticals is antibody-drug conjugates (ADCs). Here, we developed a new modular strategy for the generation of cytotoxic bioconjugates, containing a homodimer of targeting protein and two highly potent anticancer drugs with distinct mechanisms of action. Instead of antibody, we applied human fibroblast growth factor 2 (FGF2) as a targeting protein. We produced a conjugate of FGF2 with either monomethyl auristatin E (MMAE) or α-amanitin (αAMTN) as a cytotoxic agent and subsequently applied a sortase A-mediated ligation to obtain a dimeric conjugate containing both MMAE and αAMTN. The developed method ensures site-specific conjugation and a controlled drug-to-protein ratio. We validated our approach by demonstrating that dimeric dual warhead conjugate exhibits higher cytotoxic potency against fibroblast growth factor receptor-positive cell lines than single-warhead conjugates. Our modular technology can be applied to other targeting proteins or drugs and thus can be used for preparation of different bioconjugates.