Therapeutic monoclonal antibodies (mAb) constitute a group of highly effective agents for treating various refractory diseases. Nonetheless it is challenging to achieve selective and accurate quantification of mAb in pharmaceutical matrices, which is required by PK studies. Liquid chromatography/mass spectrometry under selected reaction monitoring mode (LC/SRM-MS) is emerging as an attractive alternative to immunoassays because of the high specificity and multiplexing capacity it provides, but may fall short in terms of sensitivity, reliability and quantitative accuracy. Moreover, the strategy for optimization of the MS conditions for many candidates of signature peptides (SP) and the selection of the optimal SP for quantification remains elusive. In this study, we employed a suite of technical advances to overcome these difficulties, which include i) a nano-LC/SRM-MS approach to achieve high analytical sensitivity, ii) a high-resolution nano-LC/LTQ/Orbitrap for confident identification of candidate peptides, iii) an on-the-fly orthogonal array optimization (OAO) method for the high-throughput, accurate and reproducible optimization for numerous candidate peptides in a single LC/MS run without using synthesized peptides, iv) a comprehensive evaluation of stability of candidates in matrix using the optimized SRM parameters, v) the use of two unique SP for quantification of one mAb to gauge possible degradation/modification in biological system and thus enhancing data reliability (e.g. rejection of data if the deviation between the two SP is greater than 25%) and vi) the utilization of purified target protein as the calibrator to eliminate the risk of severe negative biases that could occur when a synthesized peptide is used as calibrator. To show a proof of concept, this strategy is applied in the quantification of cT84.66, a chimeric, anti-CEA antibody, in preclinical mouse models. A low detection limit of the mAb down to 3.2 ng/mL was achieved, which is substantially more sensitive than established immunoassay methods for anti-CEA antibodies. The quantitative method showed good linearity (within the range of 12.9 ng/mL to 32.3 µg/mL in plasma), accuracy and precision. Additionally, the ultra-low sample consumption (2 µL plasma per preparation) permits the acquisition of an entire set of time course data from the same mouse, which represents a prominent advantage for PK study using small-animal models. The developed method enabled an accurate PK investigation of cT84.66 in mice following intravenous and subcutaneous administrations at relatively low doses over an extended period of time. The strategy employed in this study can be easily adapted to the sensitive and accurate analysis of other mAb and therapeutic proteins.

Exposure-response relationships for efficacy were inconsistent across exposure metrics; model-predicted cycle 1 C showed the strongest exposure-response trend. The Q1 subgroup based on model-predicted cycle 1 C had numerically similar or better OS and PFS versus control following covariate adjustment. The approved T-DM1 dose (3.6 mg/kg every 3 weeks) has a positive benefit-risk ratio versus control, even for the T-DM1 Q1 subgroup.

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ABSTRACTvc-MMAE antibody–drug conjugates (ADCs) consist of a monoclonal antibody (mAb) covalently bound with a potent anti-mitotic toxin (MMAE) through a protease-labile valine-citrulline (vc) linker. The objective of this study was to characterize the pharmacokinetics (PK) and explore exposure–response relationships of eight vc-MMAE ADCs, against different targets and for diverse tumor indications, using data from eight first-in-human Phase 1 studies. PK parameters of the three analytes, namely antibody-conjugated MMAE (acMMAE), total antibody, and unconjugated MMAE, were estimated using non-compartmental approaches and compared across the eight vc-MMAE ADCs. Relationships between analytes were assessed by linear regression. Exposure–response relationships were explored with key efficacy (objective response rate) and safety (Grade 2+ peripheral neuropathy) endpoints. PK profiles of acMMAE, total antibody and unconjugated MMAE following the first dose of 2.4 mg/kg were comparable across the eight ADCs; the exposure differences between molecules were small relative to the inter-subject variability. acMMAE exposure was strongly correlated with total antibody exposure for all the eight ADCs, but such correlation was less evident between acMMAE and unconjugated MMAE exposure. For multiple ADCs evaluated, efficacy and safety endpoints appeared to correlate well with acMMAE exposure, but not with unconjugated MMAE over the doses tested. PK of vc-MMAE ADCs was well characterized and demonstrated remarkable similarity at 2.4 mg/kg across the eight ADCs. Results from analyte correlation and exposure–response relationship analyses suggest that measurement of acMMAE analyte alone might be adequate for vc-MMAE ADCs to support the clinical pharmacology strategy used during late-stage clinical development.