Project abstract/summary In the proposed research, GlycoT will expand its cutting-edge chemoenzymatic platform by further optimizing and streamlining a one-pot single-zyme glycan-specific conjugation technology, that can be applied to a wide variety of application. Conjugation of various functional molecules to antibodies are frequently used for a wide variety of applications within the life science sectors, such as fluorescent labeled antibodies for the detection and imaging, antibody-drug conjugate (ADC) for cancer therapy, LYTAC for targeted degradation. The most exemplified application of antibody conjugation is the development of ADC therapeutics. For approved 15 ADCs and others in clinical trials, the payloads have been mainly conjugated to antibody by non-specific random linkage to either cysteine or lysine, resulting in heterogenous ADC regioisomers, with varied antigen affinity, aggregation potential, serum half-life, and other limitation. As a result, site-specific ADCs with improved pharmacokinetics, and enhanced therapeutic index have been developed. Among different approached to generate site-specific ADCs, remodeling of Fc-glycan on the conserved Asn-297 position to generate Fc-glycan specific ADC is particularly attractive. The use of the galactosyltransferase (GalT) mutants capable of accommodating modified UDP-Gal derivatives as the donor substrates has enabled the incorporation of a selected tag at the Fc glycans for subsequent site-specific conjugation with modified cytotoxic agents. This technology route has been adopted by several clinical stage companies. However, the GalT mutant is not very efficient and can only transfer azide or keto based small Gal-GDP derivative. In contrast, another endoglycosidase-based one-pot transglycosylation method has overcome such limitations. This convergent approach combined two key enzymatic steps, deglycosylation of the antibody, and subsequent attachment of a tagged disaccharide, in the same reaction system, with a single endoglycosidase like EndoS2 from S. pyogenes. This one-pot platform has been exclusively licensed by GlycoT. The core enzyme EndoS2 is highly efficient and can transfer azido disaccharide modified with single or multiple extended linkers, as demonstrated in the proof-of-concept studies. In the proposed Phase 2 R&D, GlycoT will streamline this amazing bioconjugation technology by pursuing the following five specific aims: Aim 1. Scale-up and optimization of the synthesis of disaccharides oxazolines, Aim 2. Combability of single-enzyme transglycosylation with engineered human IgG, Aim 3. Design and evaluate EndoS2 fusion enzyme to further simplify the glycoengineering process, Aim 4. Streamline the whole process to synthesize glycan-specific ADC, Aim 5. In vivo evaluation of glycan-specific ADCs. With the successful execution of this R&D plan, for the academic researchers, we can provide convenient, highly efficient, and robust labeling kits for site-specific conjugation of...