Chemically Programmed Fc Protein as Antibody Mimetic to Targeting Carbonic Anhydrase IX

Abstract

Chemically programmed antibody (cPAb) is a type of small molecule-antibody conjugate that relies on the conjugated chemical moiety for specific targeting. Although it is not as clinically successful as its comparator antibody-drug conjugate (ADC), cPAb is of great interest for rapidly developing economically attractive antibody mimetics to reduce the immunotherapeutic cost. While there are some reports on chemical approaches to develop cPAbs, the sitespecific and bio-orthogonal conjugation of small-molecule ligands to an antibody including its fragment remains an ongoing challenge. Here, we describe a generalizable method for the construction of Fc-based cPAb by site-specifically conjugating the acetazolamide (AAZ) ligands (with high affinity to the tumorassociated antigen carbonic anhydrase IX) to a human IgG1-derived Fc protein in a two-step bio-orthogonal conjugation: sortase A-mediated terminal azidation and click reaction. The resulting cPAb exhibits high specificity and avidity to the cell-surface antigen carbonic anhydrase IX of renal cancer SK-RC-52 cells. In addition, the cPAb retains the IgG recycling function, reflected by a prolonged circulation half-life (∼20 h) in mice. Cell-based bioassays also reveal that cPAb can mediate the antibody-dependent cellular cytotoxicity (ADCC) to tumor cells in a fashion dependent on glycosylation status. Our results demonstrate that cPAb can mimic antibodies in aspects of both targeting capability and effector functions, showing potential as a class of immuno-therapeutics.