Tumor-Targeting Antibody–Anticalin Fusion Proteins for in Vivo Pretargeting Applications

Abstract

Pretargeting approaches rely on the injection of bispecific antibodies capable of recognizing both an accessible disease marker and a small ligand, which is typically administered at a later stage and which serves as delivery vehicle for a payload for imaging or therapy applications. In the oncology field, pretargeting strategies have exhibited extremely promising biodistribution results and in vivo selectivity, but have often relied on the cumbersome preparation of multispecific antibodies by chemical conjugation techniques. Here, we describe the design, production, and characterization of a novel class of bispecific multivalent antibody products, which contain both antibody fragments and an anticalin moiety for the simultaneous recognition of tumor-associated antigens and a small organic molecule. Anticalins are derivatives of the naturally occurring binding proteins lipocalins, which have been engineered to recognize a target molecule with high affinity. In particular, we produced and compared in vitro and in vivo different fusion proteins, which contained the anticalin FluA that selectively recognizes various different fluorescein derivatives and the F8 antibody specific to the alternatively spliced EDA domain of fibronectin (a marker of tumor angiogenesis). The selective accumulation of the most promising fusion-protein scFv(F8)-FluA-scFv(F8) on solid tumors and simultaneous binding of fluorescein derivatives could be visualized in vivo using a fluorescein-near-infrared fluorescent dye conjugate, confirming the potential of antibody-anticalin fusion proteins for pretargeting applications.