Summary: Antibody drug conjugate (ADC) immunogenicity is a growing concern due to the increased interest in immune- stimulating drug delivery technology. A recent study by Novartis demonstrated that 14 out of 14 patients dosed with an experimental immune-stimulatory ADC developed a measurable anti-drug antibody (ADA) response. ADA negatively affects treatment by neutralizing drugs, increasing drug clearance, and causing severe allergic reactions. There is an urgent need for an effective method of conjugating payload to antibodies while minimizing the development and the effects of ADAs. We have developed an ADC bioconjugation technology that employs the conserved Q295 residue (continued funding provided by NIH 1RO1GM140026-O1A1) that we believe will mitigate many of these ADA concerns. The goal of this proposal is to demonstrate that this technology reduces Fc-gamma uptake in antigen presenting cells (APCs) and masks the drugs from binding to ADAs by hiding them in a sterically occluded hydrophobic pocket on the Fc domain. This goal will be achieved through the two aims. Aim #1 focuses on developing NMR and fluorescence-based tools to demonstrate that ADC payloads at the Q295 site are constrained within the sterically occluded IgG1 hydrophobic pocket. To this end we propose conjugating model payloads to the Q295 site using various linkers (such as short alkyl, peptide, and PEG) and determining their proximity to the hydrophobic pocket using two experimental methods: 1) a proximity induced fluorescent quenching assay using Oregon Green 488, due to its ability to self-quench and 2) F-19 protein NMR using a trifluoromethyl probe, due to its high sensitivity and low background level in biological samples. Aim #2 focuses on demonstrating that payloads attached to the sterically occluded Q295 position have reduced ADA binding and reduced potential for eliciting immunogenicity caused by Fc-gamma mediated uptake of ADCs. Achievement of the aims outlined herein will result in an antibody conjugation technology that exhibits a reduced risk of ADA and may be useful for the design of immune-stimulating antibody conjugates.