Highly regioselective remote hydroxylation of a natural product scaffold is demonstrated by exploiting the anchoring mechanism of the biosynthetic P450 monooxygenase PikCD50N-RhFRED. Previous studies have revealed structural and biochemical evidence for the role of a salt bridge between the desosamine N,N-dimethylamino functionality of the natural substrate YC-17 and carboxylate residues within the active site of the enzyme, and selectivity in subsequent CH bond functionalization. In the present study, a substrate engineering approach was conducted that involves replacing desosamine with varied synthetic N,N-dimethylamino anchoring groups. We then determined their ability to mediate enzymatic total turnover numbers (TTN) approaching or exceeding that of the natural sugar, while enabling ready introduction and removal of these amino anchoring groups from the substrate. The data establish that the size, stereochemistry and rigidity of the anchoring group influence the regioselectivity of enzymatic hydroxylation. The natural anchoring group desosamine affords a 1:1 mixture of regioisomers while synthetic anchors shift YC-17 analog C-10/C-12 hydroxylation from 20:1 to 1:4. The work demonstrates the utility of substrate engineering as an orthogonal approach to protein engineering for modulation of regioselective C-H functionalization in biocatalysis.

Passive immunization with monoclonal antibody TA99 targeting melanoma differentiation antigen tyrosinase-related protein-1 (Tyrp1; gp75) and active immunization with plasmid DNA encoding altered Tyrp1 both mediate tumor immunity in the B16 murine melanoma model. We report here that TA99 enhances Tyrp1 DNA vaccination in the treatment of B16 lung metastases, an effect mediated by immunologic mechanisms as Tyrp1 has no known role in regulating tumor growth. TA99 is shown to increase induction of anti-Tyrp1 CD8+T-cell responses to DNA vaccination against Tyrp1 as assessed by IFN-; ELISPOT assays. Immunohistochemistry studies reveal that TA99 localizes rapidly and specifically to B16 lung nodules. Augmentation of T-cell responses is dependent on the presence of tumor as well as on activating Fc receptors. Furthermore, TA99 enhances DNA vaccination against a distinct melanoma antigen, gp100(pmel17/silver locus), improving antitumor efficacy, augmenting systemic CD8+ T-cell responses to gp100, and increasing CD8+ T-cell infiltration at the tumor site. Epitope spreading was observed, with CD8+ T-cell responses generated to Tyrp1 peptide in mice receiving gp100 DNA vaccination in the presence of TA99. Finally, we show that TA99 improves therapeutic efficacy of DNA vaccination combined with adoptive T-cell transfer in treatment of established subcutaneous B16 melanoma. In conclusion, TA99 enhances DNA vaccination against both the target antigen Tyrp1 and a distinct melanoma antigen gp100 in an Fc receptor-dependent mechanism, consistent with enhanced cross-presentation of tumor-derived antigen. Monoclonal antibodies should be tested as vaccine adjuvants in the treatment of cancer. [Cancer Res 2008;68(23):9884-91]