Abstract
Trastuzumab emtansine (T-DM1, brand name: Kadcyla®) is the first FDA-approved antibody-drug conjugate (ADC) for metastatic human epidermal growth factor receptor 2 positive (HER2+) breast cancer. It consists of three components: trastuzumab, an anti-HER2 monoclonal antibody, maytansinoid (DM1) as a cytotoxic drug, and maleimidomethyl cyclohexane-1-carboxylate (MCC) as a linker. In particular, the MCC linker is known as a non-cleavable linker and has a feature of being conjugated to DM1 by a covalent thioether bond. In this study, we developed an immuno-affinity capture liquid chromatography-mass spectrometric (LC-MS/MS) assay for quantifying the antibody-conjugated drug (acDrug) component of T-DM1. To quantify acDrug, desulfurated DM1 was prepared using a chemical desulfuration pretreatment and quantified as an acDrug. A quadratic regression (weighted 1/concentration), with equation y = ax2 + bx + c, was used to fit the calibration curves over the concentration range of 17.09~1709.44 ng/mL for the acDrug of T-DM1. The quantification run met the in-house acceptance criteria of ±25% accuracy and precision values for the quality control (QC) samples. In conclusion, an immuno-affinity capture LC-MS/MS assay was successfully developed to quantify acDrug of T-DM1 and applied to evaluate in vitro plasma linker stability and preclinical pharmacokinetic (PK) study in rats. This assay could be helpful when applied to other ADCs with the same linker-cytotoxic drug platform.
Highlights
Antibody-drug conjugate (ADC) is a promising biopharmaceutical consisting of three components: a monoclonal antibody, a linker, and cytotoxic drugs [1,2,3,4,5,6,7,8,9].ADCs undergo receptor-mediated endocytosis after reaching target cells through a specific antigen-antibody response
ADCs are degraded by lysosomal enzymes, and cytotoxic drugs are released [10]
We developed an immuno-affinity capture LC-MS/MS assay using previously reported chemical desulfuration pretreatment [19] to quantify antibody-conjugated drug (acDrug) in rat preclinical PK samples even for trastuzumab emtansine (T-DM1, brand name: Kadcyla® ), a representative ADC with non-cleavable linkers
Summary
Antibody-drug conjugate (ADC) is a promising biopharmaceutical consisting of three components: a monoclonal antibody, a linker, and cytotoxic drugs (payloads) [1,2,3,4,5,6,7,8,9]. We developed an immuno-affinity capture LC-MS/MS assay using previously reported chemical desulfuration pretreatment [19] to quantify acDrug in rat preclinical PK samples even for trastuzumab emtansine (T-DM1, brand name: Kadcyla® ), a representative ADC with non-cleavable linkers. T-DM1 is the first FDA-approved ADC to treat metastatic human epidermal growth factor receptor 2 (HER2)-positive breast cancer and consists of the monoclonal antibody trastuzumab, maleimidomethyl cyclohexane-1carboxylate (MCC) as a non-cleavable linker, and maytasinoid as a payload [20]. This developed assay was applied to evaluate in vitro plasma linker stability and preclinical PK studies in rats. This is the first approach to quantitatively evaluate the acDrug of T-DM1 in vivo rat PK samples
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