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.