An Engineered Cysteine-Modified Diabody for Imaging and Treatment of Activated Leukocyte Cell Adhesion Molecule (ALCAM)-Positive Tumors

Activated leukocyte cell adhesion molecule (ALCAM) is a cell-surface glycoprotein upregulated in many malignancies, including pancreatic adenocarcinoma and colorectal carcinoma, which has shown promise preclinically as a therapeutic target. By facilitating the noninvasive assessment of tumor phenotype in vivo, molecular imaging agents can aid in the development, evaluation, and implementation of molecularly targeted therapeutics. The present work describes the production and in vitro characterization of a fully human anti-ALCAM cysteine-modified diabody (CysDb), and the in vivo evaluation of two anti-ALCAM CysDb-based positron emission tomography (PET) radiotracers. A CysDb is a bivalent antibody fragment comprised of two, cross-paired single chain variable fragments (scFv), which are covalently linked by a disulfide bond that forms between engineered cysteines on the C-termini. Importantly, these residues are located away from the antigen-binding domains, and following mild reduction, the sulfhydryl groups of the cysteines become available for modification with cargo that possesses a reactive maleimide group. An existing anti-ALCAM scFv derived from a phage display library was reformatted by genetic engineering to generate an anti-ALCAM CysDb. SDS-PAGE, size exclusion chromatography, and mass spectrometry analyses confirmed production of a covalent scFv dimer, and flow cytometry and immunofluorescence microscopy binding studies showed specific binding of CysDb to ALCAM-positive cell lines. To generate a radiotracer for use with PET, the chelator DOTA was conjugated to random lysine residues for subsequent labeling with 64Cu. 64Cu-DOTA(amide)-CysDb demonstrated specific in vivo targeting and imaging of ALCAM-positive tumors in two mouse models of pancreatic adenocarcinoma and a mouse model of colorectal carcinoma. The potential for site-specific modification was explored by coupling a variety of maleimide-containing moieties, including DOTA, to reduced CysDb. Site-specific conjugation of DOTA to the C-terminal cysteines was demonstrated by mass spectrometry, and 64Cu-DOTA(thioether)-CysDb was found to specifically target and image ALCAM-positive tumors in a mouse model of colorectal carcinoma. Successful in vivo imaging of ALCAM with CysDb-based PET radiotracers suggests that further investigation of ALCAM as an imaging biomarker is warranted. Furthermore, this work demonstrates site-specific modification of CysDb, indicating that this antibody format could be a good alternative when random modification of a diabody results in a significant loss of antigen-binding activity.