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Lead Candidate Selection of Her-2 Antibody-Drug Conjugates: Determination of Higher Order Structure Using Forced Degradation and Intrinsic Fluorescence
CASSS AT Europe 2020 -- Antibody-drug conjugates (ADCs) represent a powerful weapon in the treatment of cancer. These combination biopharmaceuticals work by specifically targeting a tumour antigen allowing the delivery of a toxic payload to inhibit cell growth and induce tumour cell death. There are three structural components to an ADC: (1) a monoclonal antibody (mAb) that selectively binds to a tumour antigen expressed by the target cell; (2) the chemical linker and (3) the potent cytotoxic drug. With cysteine conjugation, the linker-drug combination can be attached to the mAb via free thiol groups by the reduction of intermolecular disulphide bonds (Trastuzumab: anti-Her2) or in a site directed manner (Thio-Trastuzumab LC V206C). Irrespective of the site of conjugation, there is a potential for the addition of the linker-drug to destabilise the mAb higher order structure (HOS). Such destabilisation makes the ADC more prone to aggregation. Excitation of proteins at 280nm results in a fluorescence emission spectra due to the aromatic amino acid content. The major component of the intrinsic fluorescence (IF) emission spectrum is due to tryptophan; moreover, the tryptophan fluorescence is very sensitive to the solvent polarity and the local protein environment. As tryptophan residues generally reside in the hydrophobic core of a protein, as it partially unfolds there is ingress of water and resulting in a shift to longer emission wavelengths (red-shift). Consequently, IF can be used to monitor protein HOS. In this study, IF was assessed as a high throughput technique to evaluate ADC HOS stability using different stress conditions.
