Conjugation of small molecule drugs to specific sites on the antibody molecule has been increasingly used for the generation of relatively homogenous preparations of antibody-drug conjugates (ADCs) with physicochemical properties similar or identical to those of the naked antibody. Previously a method for conjugation of small molecules to glycoproteins through existing glycans by using an engineered glycotransferase and a chemically reactive sugar as a handle was developed. We used this method with some modification to generate  ADCs from several human monoclonal antibodies (mAbs) including one, m860, which we identified from a human naïve phage display Fab library by panning against the extracellular domain of human HER2. M860 bound to cell surface-associated HER2 with affinity comparable to that of Trastuzumab (Herceptin), but to a different epitope. The m860ADC was generated by enzymatically adding a reactive keto-galactose to m860 using an engineered glycotransferase and conjugating the reactive m860 to aminooxy auristatin F. It exhibited potent and specific cell-killing activity against HER2 positive cancer cells, including trastuzumab-resistant breast cancer cells. We also generated several ADCs using PBD dimers as a payload which exhibited very potent killing activity (IC50 ~ 5 pM) against cancer cell lines including some expressing CD56.  These ADCs were very stable in human serum and didn’t change their specific killing activity even after a month at physiological temperature. Our results validate the keto-galactose/engineered glycotransferase method for generation of functional ADCs, which could potentially also be used for preparation of ADCs targeting other diseases.

Antibodies conjugated through glycans to small molecule drugs: Stability and specific killing of cancer cells

251st National Meeting (2016)

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