Technology/ Title:Tri-mannosyl Antibody: A Versatile Payload ADC Platform

Summary of Invention
We have developed a high efficiency glycoengineering technology by using rabbit GnT-I (N-Acetyl glucosamine transferase I) and rat GnT-II (N-Acetyl glucosamine transferase II) to conjugate a tri-mannosyl core antibody and produce a novel format ADC. To validate our platform, Herceptin was sequentially digested to generate a tri-mannosyl Herceptin antibody. The tri-mannosyl ADC with DAR4 (drug-to-antibody ratio) is generated by converting the tri-mannosyl antibody to a tri-mannosyl -4GlcNAz antibody and then the Dibenzocyclooctyne (DBCO) -(PEG)4-DM1 was linked to the terminal GlcNAz group in the antibody by strain-promoted azide-alkyne click chemistry (SPAAC) reaction. Our results demonstrate that the conversion efficiency of the ADC is over 98%. We further proved the feasibility to generate a dual payload ADC with DAR4 from a tri-mannosyl antibody. Our results show that the dual payload ADC is generated with the conversion efficiency over 90%. The tri-mannosyl Herceptin ADC product not only has a similar KD to that of commercial Kadcyla, but also has cytotoxic activities to Her2/Neu medium-expression cell line HCC-1954. These studies suggest that rabbit GnT-1 and rat GnT-2 have very good potential to develop a dual payload and site-specific ADC platform when a tri-mannosyl antibody is used as a start material.

Advantages when compared to the existing technologies
1. Correct light chain and heavy chain pairing.
2. Longer half-life.
3. Target cancer cell-dependent T cell activation.
4. Better anti-cancer efficacy compared to the short half-life fragment BsAb and its parental monoclonal antibody.
5. High production yield comparable to its parental mAb.
6. Low Immunogenicity in rodent.