Designing the Aplysia punctata Arginine-Depleting Enzyme for Tumor Targeting.

l-Amino acid oxidases (LAAO) deaminate amino acids to α-keto acids and generate hydrogen peroxide, a reactive oxygen species (ROS) with potential value for cancer therapy. We recombinantly expressed the LAAO from Aplysia punctata, called APIT (Cuvier 1803). The resulting wild-type APIT (APITwt) was conjugated to polyethylene glycol (APIT-PEG). Furthermore, an APIT mutant with an affibody targeting the human epidermal growth factor receptor 2 (HER2; zHER2-APIT) was genetically engineered resulting in a binding affinity KD of ∼ 2.2 nM to the HER2 receptor ectodomain. Further, we evaluated if the APIT and tumor-targeted APIT can be used as an APIT-drug conjugate by covalently amidating the lysine residues on the protein surface. However, for the HER2-targeted APIT, the affibody contains lysines as well, and amidation of these lysines could have impaired the affibody's affinity to the HER2 receptor. Therefore, we designed a lysine-free variant of the tumor-targeting part of zHER2-APIT using an in silico mutation analysis, suggesting the replacement of the lysines of the affibody by arginine or alanine. This new variant is referred to as zHER2(K-del)-APIT. To simulate a covalent drug loading to APIT and the targeting constructs, we attached biotin by amidation. Biotin-zHER2(K-del)-APIT successfully allowed binding to HER2-positive but not HER2-negative cells in vitro. The biodistribution of these novel constructs was tested in xenografted mice with a HER2-positive and negative tumor in each animal. The zHER2(K-del)-APIT lost its ability to target HER2-positive tumors despite the in vitro data suggesting otherwise. The zHER2-APIT accumulated within the HER2-positive tumors but not in the negative tumors. APIT-PEG had increased uptake in HER2-positive and negative tumors compared to APITwt, which can be attributed to a prolonged serum half-life achieved by PEGylation, due to the absence of any tumor-targeting effect. These biodistribution studies point to HER2-targeting LAAOs for cancer therapy and PEGylation increasing tumor accumulation.