Site-Specific Chemical Modification of a Cytokine Mimic for Small Molecule-Based Tumor Targeting

The targeted delivery of bioactive proteins, such ascytokines,for cancer immunotherapy approaches mostly relies on antibodies orantibody fragments. However, fusion proteins may display low tissuepenetration due to a large molecular size. Small molecule ligandswith high affinity toward tumor-associated antigens provide a promisingalternative for the selective delivery of cytokines to tumor lesions.We developed a one-pot procedure for the site-specific thiazolidineformation between an aldehyde bearing small molecule and the in situ generated N-terminal cysteine of a bioactive protein.Thereby, neoleukin-2/15 (Neo-2/15), a computationally engineered interleukin-2and -15 mimic, was chemically conjugated to acetazolamide plus, apotent carbonic anhydrase IX (CAIX) ligand. The conjugate retainedthe biological activity of Neo-2/15 and revealed its ability to accumulatein renal cell carcinoma (SK-RC-52) xenografts upon systemic intravenousadministration. The results highlight the potential of small moleculetargeting moieties to drive the accumulation of a protein cargo tothe respective disease site while conserving the small construct size.